List of Abstracts – updated 4 April 2014

Transcription

TABLE OF CONTENTS
SESSION 1: KEYNOTE 1
Solving nanoscale structures using focussed electron beams
Joanne Etheridge
1 SESSION 2: BEYOND STRUCTURE 1
Twists and turns: the lessons hidden in disorder
Paul J. Low, Dmitry S. Yufit and Judith A.K. Howard
INVITED SPEAKER
3 4 Biosensors to probe the nexus between protein conformations and cell functionality
Danny M. Hatters, Rebecca Wood, Sevgi Irtegun, Yasmin Ramdzan and
Angelique Ormsby
INVITED SPEAKER
Beyond structure with a WOMBAT, applications of neutron powder diffraction
to planetary science
Helen E. Maynard-Casely, Andrew J. Studer, Max Avdeev, Helen E.A. Brand
and Kia S. Wallwork
INVITED SPEAKER
2 Protein nanotechnology: Approaches to generating useful nanomaterials using
protein tectons
Juliet Gerrard
INVITED SPEAKER
5 SESSION 3: MATHIESON MEDAL LECTURE
Medal Presentation and Lecture
6 SESSION 4: KEYNOTE 2
Crystallography without crystals: Order within disorder
Andrew Goodwin
7 SESSION 5: HOT STRUCTURES IN BIOLOGY
Spring-hammer mechanism of metal ion binding and release by the Streptococcus
pneumoniae substrate-binding protein PsaA
Rafael M. Couñago , Zhenyao Luo , Miranda P. Ween , Stephanie L. Begg ,
Megha Bajaj , Johannes Zuegg , Megan L. O’Mara , Matthew A. Cooper ,
Alastair G. McEwan , James C. Paton , Christopher A. McDevitt and
Bostjan Kobe
INVITED SPEAKER
8 Structural insights into cell death
Peter Czabotar
INVITED SPEAKER
CRYSTAL29 – 29th Conference of SCANZ - 2014
9 i
The Signal Recognition Particle RNA and its multi functions
10 Felix Voigts-Hoffmann, Nikolaus Schmitz, Kuang Shen, Shu-ou Shan, Nenad Ban
and Sandro F. Ataide
Exploring the conformational flexibility of human IgE with a inhibitory Fab fragment
Nyssa Drinkwater, Ben Cossins, Anthony Keeble, Andrew Beavil,
James McDonnell, Alistair Henry and Brian Sutton
Insights into the recruitment of the PAN2-PAN3 deadenylase complex to miRNA
targets by the GW182/TNRC6 proteins
Mary Christie, Andreas Boland, Eric Huntzinger, Oliver Weichenrieder and
Elisa Izaurralde
Structural studies of the cavin proteins provide new insights into the mechanisms of
caveolae formation
Oleksiy Kovtun, Vikas Tillu, WooRam Jung, Natalya Leneva, Nick Ariotti,
Kirill Alexandrov, Rob Parton and Brett Collins
11 12 13 SESSION 6: CRYSTAL ENGINEERING
Exploiting redox-activity in metal-organic frameworks and porous coordination
polymers for functional properties
Deanna M. D’Alessandro, Thomas B. Faust, Carol Hua, Chanel Leong,
Weibin Liang, and Pavel M. Usov
INVITED SPEAKER
14 Flexible crystals: Stretching the boundaries of a single crystal
Jack K. Clegg, John C. McMurtrie, Anna Worthy, Michael Pfrunder and
Aidan Brock
INVITED SPEAKER
15 The presence of mixed valence rare earths inRMn2X2compounds
16 Shane Kennedy, Jianli Wang, Stewart Campbell, Michael Hofmann and
Shixue Dou
Halogen-bond mediated crystal engineering of metal complexes
17
Michael Pfrunder, Llew Rintoul, Dennis Arnold, Madeleine Shultz, Jack Clegg and
John McMurtrie
Spin crossover induced molecular mosaic patterning generated in a 3D Porous MOF
Natasha F. Sciortino, Suzanne M. Neville, Gregory J. Halder, Keith S. Murray,
Boujemaa Moubaraki, Jean-François Létard, Cameron J. Kepert
18 Combined neutron scattering and DFT modelling studies of functional materials
Samuel G. Duyker, Vanessa K. Peterson, Gordon J. Kearley, Anibal J. RamirezCuesta and Cameron J. Kepert
19 SESSION 7: BEYOND STRUCTURE 2
Let the powers combine: hybrid methods in structural biology.
Jane R Allison
INVITED SPEAKER
20 Reverse electrostatic complementarity based on the crystal structure of the pure
protease inhibitor E64c
Ming Wen Shi and Simon Grabowsky
21 ii
Crystal structures of ancestral amino acid binding proteins reveal protein specialization
can occur through conformational selection
Colin Jackson and Benjamin Clifton
INVITED SPEAKER
22 Crystal structures of genomic island components from Australian A. Baumannii strains
Bhumika Shah, Stephen Harrop, Ian Paulsen and Bridget Mabbutt
23 X-ray wavefunction refinement – introduction, examples, validation
Simon Grabowsky, Magdalena Woińska, Joanna M. Bąk and Dylan Jayatilaka
24 INVITED SPEAKER
SESSION 8: MATERIALS AND METALS
Using hydrogels to control crystallization: synthesizing next generation implant
materials
Kathryn M. McGrath
INVITED SPEAKER
25 Probing electrochemistry and crystal structure simultaneously
Neeraj Sharma
INVITED SPEAKER
26 First observation of intersite valence transitions involving 4d and 5d ions, as revealed
by high-pressure x-ray and neutron diffraction
Chris D. Ling, Zixin Huang, Brendan J. Kennedy Wojciech Miiller and
Max Avdeev
27 Critical applications of high absolute accuracy XAFS measurements to low-energy
inelastic electron scattering
Jay D. Bourke and Christopher T. Chantler
28 Investigation and control of periodic microphases in composites of block copolymers
and ionic liquids
Kevin Jack, Thomas Bennett, Kristofer Thurecht and Idriss Blakey
29 SESSION 9: BRAGG MEDAL LECTURE
Targetting multidrug resistance in Neisseria: A structural approach
Alice Vrielink
31 iii
SESSION 11: DRUG TARGETS AND INHIBITOR DEVELOPMENT
Structural diversity among autotransporter proteins
Begoña Heras, Jason J Paxman, Makrina Totsika, Kate M Peters and
Mark A Schembri
INVITED SPEAKER
32 Crystal structure and immunological properties of the first annexin from Schistosoma
mansoni
Andreas Hofmann, Chiuan Yee Leow5, Charlene Willis, Asiah Osman, Lyndel
Mason, Anne Simon6, Brian J. Smith7, Robin B. Gasser and Malcolm K. Jones
33 The structure of a chorismate-utilizing enzyme from Mycobacterium tuberculosis
supports the possibility of developing a “magic shot-gun” rather than a “magic bullet”
34 Genevieve Evans, Ghader Bashiri, Jodie M. Johnston, Esther M.M. Bulloch,
Alexandra Manos-Turvey, Richard J. Payne, Edward N. Baker and J. Shaun Lott
Bacterial disarmament: DSB proteins as anti-virulence targets
Róisín M. McMahon, Philip M. Ireland, Kieran Rimmer, Lakshmanane
Premkumar, Mitali Sarkar-Tyson, Craig Morton, Martin J. Scanlon and
Jennifer L. Martin
35 Design, development and validation of galectin-3-specific inhibitors
Khuchtumur Bum-Erdene and Helen Blanchard
36 Tetrahydrocarbazoles: a future broad-spectrum antibiotic?
Zhou Yin, Louise R. Whittell, Jennifer L. Beck, Michael J. Kelso and
Aaron J. Oakley
37 INVITED SPEAKER
SESSION 12: CHEMICAL CRYSTALLOGRAPHY
Laue neutron diffraction: Applications in chemical crystallography.
David R. Turner, Gregory S. Hall, Adrian J. Emerson, Laura J. McCormick,
Ross O. Piltz and Alison J. Edwards
INVITED SPEAKER
38 Targeting multi-step spin crossover in flexible coordination polymers
Natasha F. Sciortino, Florence Ragon, Boujemaa Moubaraki, Keith S. Murray,
Jean-François Létard, Cameron J. Kepert and Suzanne M. Neville
39 Versatile nanoballs – multiple packings and multiple properties
Stuart R. Batten
40 Resolution, interpretation and modelling in chemical crystallography – critical analysis –
furthering chemistry!
41 Alison J. Edwards
Local order in wüstite. Fe1-xO, using a PDF approach
T.R. Welberry, D.J. Goossens and A.P. Heerdegenr
What will the new Volume of the International Tables for Crystallography be about?
Volume I: XAS, Eds CT Chantler, B Bunker, F Boscherini
Chris T. Chantler
42 43 iv
SESSION 13: METHODS IN STRUCTURAL BIOLOGY
Fragment-based molecular replacement with Phaser
Airlie J. McCoy and Randy J. Read
INVITED SPEAKER
44 Structural insights into the role of the cyclic backbone in a squash trypsin inhibitor
Norelle L. Daly, Louise Thorstholm1, Kathryn P. Greenwood, Gordon J. King,
K. Johan Rosengren, Begoña Heras, Jennifer L. Martin and David J. Craik
INVITED SPEAKER
45 Validation of ligands in X-ray crystal structures
Alpeshkumar K. Malde, and Alan E. Mark
46 The structure of the megadalton magnesium chelatase assembly
Anthony P. Duff, Shabnam T. Tabrizi, Artur Sawicki, Anna V. Sokolova,
Kathleen Wood, Tom Joss, Alison M. Kriegel, Robert D. Willows,
47 First structure of a non-processive pectin methylesterase (from Aspergillus niger):
comparison to processive orthologues from plants and bacteria
Geoffrey B. Jameson, Lisa M. Kent, Gillian E. Norris, Laurence D. Melton,
Davide Mercadante and Martin A.K. Williams
48 SESSION 14: MEMBRANE PROTEINS
Structure, function, and inhibitors of the acid-gated Helicobacter pylori urea channel,
an essential component for acid survival
Hartmut Luecke
INVITED SPEAKER
49 Structures of the full-length bitopic membrane protein CYP51 from Saccharomyces
cerevisiae provide insight into substrate and drug binding and mutations affecting
antifungal susceptibility
50 Joel D.A. Tyndall, Alia Sagatova, Thomas M. Tomasiak, Mikhail V. Keniya,
Franzi U. Huschmann, Joseph D. O’Connell III, Andrew Rodruigez,
Janet Finer-Moore, Jeffrey G. MacDonald, Richard D. Cannon, Robert M. Stroud
and Brian C. Monk
Crystal structure determination of the integral membrane diacylglycerol kinase
Dianfan Li, Joseph A. Lyons, Valerie E. Pye, Lutz Vogeley, David Aragão,
Colin P. Kenyon, Syed T. A. Sha, Christine Dohert5, Margaret Aherne and
Martin Caffrey
51 A tale of three structures
Megan L. O’Mara, Karmen Condic-Jurkic, Nandhitha Subramanian,
Roisin M. McMahon and Alan E. Mark.
52 Optimisation of G protein coupled receptor expression and purification for structural
studies
53 Julia K. Archbold, Patricia M. Walden, Fabienne A. Ferreira, Matthew J. Sweet,
David E. Drew, and Jennifer L. Martin
v
SESSION 15: SCANZ 1987 PLENARY LECTURE
Membrane protein serial femtosecond crystallography using
Petra Fromme
54 SESSION 16: RISING STAR PLENARY SESSION
The protein encapsulation and delivery mechanism of ABC toxins
Jason N. Busby, Santosh Panjikar, Michael J. Landsberg, Mark R. H. Hurst
and J. Shaun Lott
The low-temperature magnetic anomaly of Ca2Fe2O5 studied by single-crystal neutron
diffraction
Josie E. Auckett, Garry McIntyre, Maxim Avdeev, Hank De Bruyn and
Chris D. Ling
The structure and dynamics of rotary ATPases
Alastair G. Stewart, Lawrence K. Lee, Mhairi Donohoe, Jessica J. Chaston and
Daniela Stock
New structural insights into receptor activation and assembly in the beta common
cytokine family
Sophie E. Broughton, Timothy R. Hercus, Tracy L. Nero, Urmi Dhagat,
Barbara J McClure, Mara Dottore, Angel F Lopez and Michael W Parker,
Structural basis of disease resistance in flax against flax rust
Thomas Ve, Simon Williams, Maud Bernoux, Ann-Maree Catanzariti,
Motiur Rahman
Insights into vibrational stark effect from X-ray charge density analysis of
supramolecular host-guest complexes
Sajesh P. Thomas, Rebecca O. Fuller, Alexandre N. Sobolev, Philip A. Schauer,
Simon Grabowsky, George A. Koutsantonis and Mark A. Spackman
55 56 57 58 59
Crystallography and climate change: The temperature-dependence of biological rates
Vic Arcus
61 POSTER SESSION
Vasorelaxant activity of Canavalia grandiflora seed lectin: A structural analysis
62 Maria Júlia Barbosa Bezerra, Ito Liberato Barroso-Neto IL, Rafael da Conceição
Simões, Bruno Anderson Matias Rocha, Francisco Nascimento Pereira-Junior,
Vinicius José Silva Osterne, Kyria Santiago Nascimento, Celso Shiniti Nagano, Plinio
Delatorre, Maria Gonçalves Pereira, Alana Freitas Pires, Alexandre Holanda
Sampaio, Ana Maria Assreuy, Benildo Sousa Cavada
Characterisation of adenylosuccinate synthetase from the fungal pathogen
Cryptococcus neoformans
63 Ross Blundell, Simon J. Williams Carl A. Morrow, Bostjan Kobe and James A. Fraser Crystallography365 and Crystals in the City: IYCr 2014 activities
Helen Maynard-Casely and Neeraj Sharma
64
vi
Solvent exchange in a hierarchically assembled metal-organic framework
Aidan J. Brock and Jack K. Clegg
Density functional calculations of electron energy loss data and inelastic mean free
paths in elemental and binary materials
65 66 Structural insights into Bak activation and oligomerisation
Jason M. Brouwer, Adeline Y. Robin, Geoff V. Thompson , Ahmad Z. Wardak,
Peter M. Colman and Peter E. Czabotar
67 Structural and dynamic studies of PPARγ agonism
Laura Marrewijk, David Marciano, Ted Kamenecka, Patrick Griffin and
John B. Bruning
68 In search of infinite solutions; the inverse problem in small-angle X-ray scattering
Lachlan W. Casey, Alan E. Mark and Bostjan Kobe
69 Structural basis of binding specificity between nuclear receprtor: importin-α and
non-classica nuclear localization signals
Chiung-Wen Chang , Rafael L.M. Couñago, Simon J. Williams , Mikael Bodén
and Boštjan Kobe
70 High accuracy calibration of a synchrotron x-ray beam using powder diffraction
71 L.J.Tantau, M.T.Islam, C.T. Chantler, N.A.Rae, A.T.Payne, C.Q.Tran and M.H.Cheah The intersection of two central cellular trafficking pathways: the binding of SNX27
to the retromer complex
Thomas Clairfeuille, Matthew Gallona, Caroline Mas, Peter J. Cullen and
Brett M. Collins
72 Structural investigations into the control of pro-apoptotic Bcl-2 family proteins
Angus D Cowan, Peter E. Czabotar and Peter M. Colman
73 Implications of the cysteine-tyrosine crosslink in cysteine dioxygenase
M. Fellner, R.J. Souness, E.P. Tchesnokov, S.M. Wilbanks and G.N.L. Jameson
74 Into the future with CIF: advances in data, metadata and meta-metadata applications
Nick Spadaccini, Doug du Boulay and Syd Hall
75 Molecular basis of signaling by Toll/Interleukin-1 receptor domain-containing
adaptors in Toll-like receptor pathways
Shane Horsefield, Thomas Ve, Bostjan Kobe.
76 Neutron study of the magnetic structures & phase transition in iron (III) phosphate, FePO4 77 Christopher J. Howard, Paul J. Saines, James R. Hester and Anthony K Cheetham Structural characterization of the mammalian CAD multienzyme complex
78 Yujung Jeon, Ian L. Ross, Michael J. Landsberg, Ben Hankamer, and Bostjan Kobe The functional and structural role of cysteine residues in Toll-like receptor signalling
adaptor MAL/ TIRAP
Peter Lavrencic, Thomas Ve, Mehdi Mobli, Bostjan Kobe
Structurally guided small molecule targeting of the insulin and Type 1 insulin-like
growth factor receptors
C.F. Lawrence, J.G. Menting, M.B. Margetts, K.E. Jarmin, K.N. Lowes,
G. Lessene, K. Lakovic and M.C. Lawrence
79 80 vii
Structural characterisation of the retromer complex and associated sorting nexins
Natalya Leneva, Suzanne Norwood, Rajesh Ghai, Nathan Cowieson,
Anthony Duff , Kathleen Wood and Brett Collins
Structural elucidation of zinc acquisition mechanisms in Streptococcus pneumoniae
and development of novel antibiotics against streptococcal diseases
Zhenyao Luo, Rafael Couñago, Alastair McEwan, Christopher McDevitt and
Boštjan Kobe
81 82 Phylogenetic mapping and structural comparison of bacterial ketol-acid reductoismeraes 83 You Lv, Luke W. Guddat, Bostjan Kobe and Mark A. Schembri
Single molecule magnetism in µ-phenolato dinuclear lanthanide complexes containing
heptadentate Schiff base ligand
Melina Nematirad, Stuart R. Batten and Keith S. Murray
Explicit and implicit data merging in crystallographic least squares
A. David Rae
84 85 Elaboration of benzoylurea inhibitors targeting pro-survival Bcl-xL
86 Michael Roy, Amelia Vom, Soo San Wan, Hong Yang, Brian Smith, Peter Colman,
Guillaume Lessene and Peter Czabotar
Crystallization and Structure characterization of the Gun4 from Chlamydomonas
reinhardtii
Shabnam Tarahi Tabrizi, Anthony Duff , Stephen J. Harrop, Arthur Sawicki,
Robert D. Willows
Multiple binding modes of isothiocyanate inhibitors of macrophage migration
inhibitory factor define structure activity relationships
Joel D.A. Tyndall, Emma S. Spencer, Edward J. Dale,Aimée L. Gommans,
Malcolm T. Rutledge,Christine T. Vo,Yoshio Nakatani, Allan B. Gamble,
Robin A. J. Smith, Sigurd M. Wilbanks,and Mark B. Hampton
A novel N-terminal domain may dictate the role of the disulfide bond protein α-DsbA2
from Wolbachia pipientis
Patricia M Walden, Premkumar Lakshmanan1, Fabian B Kurth,
Iñaki Iturbe-Ormaetxe, Maria A Halili, Julia K Archbold, Begoña Heras and
Jennifer L Martin
87 88 89
Disease resistance signaling in plant innate immune receptors, it’s all about dimerisation 91 Simon Williams, Kee Hoon Sohn, Li Wan, Maud Bernoux, Panagiotis F. Sarris,
Cecile Segonzac, Thomas Ve, Yan Ma, Simon B. Saucet, Daniel J. Ericsson,
Lachlan Casey, Xiaoxiao Zhang, Anne Coerdt, Jane Parker, Peter Dodds,
Jonathan Jones and Bostjan Kobe
Crystal structure of a flax cytokinin oxidase & interaction studies with a fungal effector
Li Wan, Markus Koeck, Simon Williams, Peter Dodds, Jeffrey Ellis and
Bostjan Kobe
Plants vs. pathogens: structural and functional studies of Arabidopsis resistance
protein SNC1 TIR domain and flax rust effector protein AvrP
Xiaoxiao Zhang, Simon Williams, Thomas Ve, Peter N. Dodds, Jeffrey G. Ellis
and Bostjan Kobe
92 93 viii
Chair: Mark Spackman, UWA
Solving nanoscale structures using focussed electron beams
Joanne Etheridge
Monash University
1
SESSION 2: BEYOND STRUCTURE 1
Chair: Geoff Jameson, Massey
Twists and turns: the lessons hidden in disorder
Paul J. Low,1 Dmitry S. Yufit2 and Judith A.K. Howard1,2
INVITED SPEAKER
1
School of Chemistry and Biochemistry, University of Western Australia,35 Stirling Highway,
Crawley, WA, Australia.
2
Department of Chemistry, South Rd, Durham University, Durham, DH1 3LE, UK.
For the synthetic chemist crystallographically determined molecular structures and optimized
molecular geometries obtained from quantum chemical protocols are powerful tools in the
elucidation of molecular structure-property relationships. However, both techniques are
generally time independent methods of structure determination, and it is apparent that
extrapolation of information from a single molecular geometry to the properties of a molecule
in fluid solution can be misleading. In this presentation attention will be turned to
organometallic acetylide complexes, which offer an interesting range of optoelectronic
properties, ranging from 2nd and higher order optical non-linearities to electronic properties
such as wire-like conductance and transistor-like behavior of interest in the construction of
single molecule electronic devices. The role of crystallographically determined structures
used in concert with DFT-based molecular geometries and spectroscopic modeling in
deriving structure-property relationships in this class of compound will be discussed, and the
intriguing hints and pointers from crystallographic structure determinations that have perhaps
been overlooked will be highlighted.
Figure 1 – A plot showing difference in spin density at the metal sites in
[{Ru(dppe)Cp*}2(µ-C≡CC6H4C≡C)]+ as a function of molecular conformation.
References
[1] Low P.J. (2013), “Twists and turns: Studies of the complexes and properties of bimetallic complexes featuring
phenylene ethynylene and related bridging ligands”, Coord. Chem. Rev. 257:1507-1532.
[2] Parthey M., Gluyas J.B.G., Schauer P.A., Yufit D.S., Howard J.A.K., Kaupp M. and Low P.J. (2013), “Refining the
interpretation of near-infrared band shapes in a polyynediyl molecular wire”, Chem. Eur. J. 19:9780-9784.
[3] Fox M.A., Le Guennic B., Roberts R.L., Brue D.A., Yufit D.S., Howard J.A.K., Manca G., Halet J.F., Hartl F.and Low
P.J. (2011) “Simultaneous bridge-localized and mixed-valence character in diruthenium radical cations featuring
diethynylaromatic bridging ligands”, J. Am. Chem. Soc. 133:18433-18446
2
Protein nanotechnology: Approaches to generating useful
nanomaterials using protein tectons
Juliet Gerrard
INVITED SPEAKER
Canterbury University, Christ Church, New Zealand
3
Beyond structure with a WOMBAT, applications of neutron powder
diffraction to planetary science
Helen E. Maynard-Casely1, Andrew J. Studer1, Max Avdeev1, Helen E.A. Brand2 and Kia S.
Wallwork1
INVITED SPEAKER
1
Australian Nuclear Science and Technology Organisation, Lucas Heights NSW 2234, Australia.
Australian Synchrotron 800 Blackburn road, Clayton, VIC 3168, Australia.
E-mail: [email protected]
2
The study of planetary science represents one to the frontiers of human endeavour. From
data returned by audacious space missions we know have a growing picture of the chemical
compositions as well as the pressure/temperature conditions of most of our planetary
neighbours. However in these alien environments we still have very little idea on the crystal
structures and behaviours of planetary-forming materials under these highly varying
conditions. One such area has been the study of ‘planetary ice’ materials. From Jupiter and
beyond in our solar system, water and other small molecular species (such as methane and
ammonia) become the dominant planetary forming materials. The intrinsic hydrogendomination of these planetary ices, makes studying these materials with laboratory powder
diffraction very challenging. Insights into their crystalline phase behaviour and the extraction
of a number of thermal and mechanical properties is only possible through diffraction studies
using either the high-flux of synchrotron x-ray sources use of the large scattering cross
section of hydrogen (deuterium) with neutron diffraction.
We have previously used the Powder Diffraction beamline at Australian Synchrotron [1] and
the ECHIDNA (High-resolution neutron powder diffraction) instrument at the OPAL reactor,
ANSTO [2] to obtain an number of new insights into the crystalline phases formed from
H2SO4/H2O mixtures under conditions relevant to the Galilean ice moons. These instruments
have enabled the discovery a new water-rich sulfuric acid hydrate form [3], improved
structural characterisation of existing forms [4] and a charting the phase diagram of this
fundamental binary system [5]. This has revealed exciting potential for understanding more
about the surface of Europa from space, perhaps even providing a window into its past.
This talk will present some of these previous results and look to the future of how the
versatile high-intensity powder diffraction instrument, WOMBAT [6], will be used for
planetary science studies.
References
[1] Wallwork K.S., Kennedy B.J. and Wang, D. (2007), “The high resolution powder diffraction beamline for the
Australian Synchrotron”, AIP Conference Proceedings, 879: 879-882.
[2] Liss K.D., et al. (2006) “Echidna - the new high-resolution powder diffractometer being built at OPAL” Physica BCondensed Matter, 385-86: 1010-1012.
[3] Maynard-Casely H.E., Wallwork K.S. and Avdeev M. (2013) “A new material for the icy Galilean moons: The
structure of sulfuric acid hexahydrate.” Journal of Geophysical Research: Planets, 118(9): 1895-1902
[4] Maynard-Casely H.E., Brand H.E.A. and Wallwork K.S. (2012) “The structure and thermal expansion of sulfuric acid
octahydrate”. Journal of Applied Crystallography, 45: 1198-1207.
[5] Maynard-Casely H.E., Brand H.E.A. and Wallwork K.S. (In Review) “The water-rich H2SO4/H2O phase diagram, a
potential marker of thermal history on Jupiter’s icy moons”
[6] Studer A.J., Hagen M.E., and Noakes T.J. (2006) “Wombat: The high-intensity powder diffractometer at the OPAL
reactor”. Physica B: Condensed Matter, 385–386, Part 2(0): 1013-1015.
4
Biosensors to probe the nexus between protein conformations and
cell functionality
Danny M. Hatters1, Rebecca Wood1, Sevgi Irtegun1, Yasmin Ramdzan1 and Angelique
Ormsby1
INVITED SPEAKER
1
Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and
Biotechnology Institute. The University of Melbourne. VIC 3010 Australia
High resolution structural methods have revolutionized our ability to understand protein
function. However the structures we see in the PDB are static and commonly in isolated
contexts. It remains a challenge to understand how protein conformation relates to their
function, their cellular localization and protein interaction networks in an intact living cell.
Our research program is centered on building new methods and approaches to define how
distinct protein conformations engage with the rest of the cell. In this talk, we discuss our
overall approaches we have used and are developing in context of three research project
vignettes. The first is our major effort to understand the process for how a protein misfolds,
subsequently aggregates into different sized aggregates, and how this process impacts on cell
physiology and health (1-3). We also discuss a strategy we devised to distinguish “open”
active conformations of the Src family kinases from “closed” inactive conformations in live
cells, as a means to probe Src signaling properties in context of the functioning networks of a
whole cell. We also discuss a new ongoing effort to quantitate how much energy a cell
invests in maintaining a healthy and folded proteome.
References
[1] Ramdzan Y.M., Nisbet R.M., Miller J., Finkbeiner S., Hill A.F. and Hatters D.M. (2010), “Conformation sensors that
distinguish monomeric proteins from oligomers in live cells”, Chem. Biol. 17:371-379.
[2] Ramdzan Y.M., Polling S., Chia C.P., Ng I.H., Ormsby A.R., Croft N.P., Purcell A.W., Bogoyevitch M.A., Ng D.C.,
Gleeson P.A. and Hatters D.M. (2012), “Tracking protein aggregation and mislocalization in cells with flow
cytometry”, Nat. Methods 9:467-470.
[3] Ormsby A.R., Ramdzan Y.M., Mok Y.-F., Jovanoski K.D. and Hatters D.M. (2013), “A platform to view Huntingtin
Exon 1 aggregation flux in the cell reveals divergent influences from chaperones hsp40 and hsp70”, J. Biol. Chem.
288:37192-37203.
5
SESSION 3: MATHIESON MEDAL LECTURE
Chair: Alice Vrielink, UWA
6
Chair: Jack Clegg, UQ
Crystallography without crystals: Order within disorder
Andrew Goodwin
Oxford University, UK
7
SESSION 5: HOT STRUCTURES IN BIOLOGY
Chair: Joel Tyndall, Otago
Spring-hammer mechanism of metal ion binding and release by the
Streptococcus pneumoniae substrate-binding protein PsaA
Rafael M. Couñago 1, Zhenyao Luo 1, Miranda P. Ween 2, Stephanie L. Begg 2, Megha Bajaj
1
, Johannes Zuegg 1, Megan L. O’Mara 1, Matthew A. Cooper 1, Alastair G. McEwan 1, James
C. Paton 2, Christopher A. McDevitt and Bostjan Kobe 1
INVITED SPEAKER
1
School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre
and Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072
2
Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of
Adelaide, Adelaide, South Australia 5005, Australia
Dynamics and flexibility are properties difficult to study by crystallography alone. We have
combined a number of approaches to define the mechanism of metal binding/release in a
bacterial substrate-binding protein. Transition row metal ions are both essential and toxic to
bacteria, which face the challenge of achieving specificity in their acquisition. In
Streptococcus pneumoniae, one of the world’s foremost bacterial pathogens, PsaA is the
substrate-binding protein of the ABC transporter responsible for manganese acquisition [1].
We have previously shown that zinc binds to PsaA, rendering bacteria susceptible to Zn
toxicity, which is in fact exploited by the host immune response [2]. We have now combined
crystallography with mutagenesis, engineering disulfide bonds, biochemistry and
microbiology to obtain a dynamic view of the binding/release process [3]. The data
collectively suggest a novel manganese-binding/release mechanism akin to a ‘springhammer’. Closing of one domain of the two-domain protein upon metal binding pushes a
metal-coordinating residue (the ‘hammer’) onto the other metal-binding residues (the ‘anvil’).
This distorts the helical turn in the segment linking the two domains (the ‘spring’). There is a
correlation between the distortion of the ‘spring’ helical turn and the ability of the protein to
bind and release metal ions. Manganese, favoring octahedral coordination, partially distorts
the helical turn and can be released during transport, while zinc, favoring tetrahedral
coordination, completely distorts the helical turn, locks the protein in a closed state and
cannot be released (explaining zinc toxicity). Our results explain the molecular basis of PsaA
specificity for manganese during competition from the more abundant zinc ions, and also
provide the foundation for structure-based drug design targeting PsaA.
References
[1] Counago R.L., McDevitt C.A., Ween M.P. and Kobe B. (2012), “Prokaryotic substrate-binding proteins as targets for
antimicrobial therapies”, Curr Drug Targets 13:1400-1410.
[2] McDevitt C.A., Ogunniyi A.D., Valkov E., Lawrence M.C., Kobe B., McEwan A.G. and Paton J.C. (2011), “A
molecular mechanism for bacterial susceptibility to zinc”, PLoS Pathog 7: e1002357.
[3] Counago R.M., Ween M.P., Begg S.L., Bajaj M., Zuegg J., O'Mara M.L., Cooper M.A., McEwan A.G., Paton J.C.,
Kobe B. and McDevitt C.A., “Imperfect coordination chemistry facilitates metal ion release in the Psa permease“,
Nature Chem Biol 10:35-41.
8
Structural insights into cell death
Peter Czabotar
INVITED SPEAKER
WEHI
9
The Signal Recognition Particle RNA and its multi functions
Felix Voigts-Hoffmann1, Nikolaus Schmitz1, Kuang Shen3, Shu-ou Shan3, Nenad Ban1 and
Sandro F. Ataide2
1
ETH Zurich (Swiss Federal Institute of Technology), Institute of Molecular Biology and Biophysics,
Zürich, Switzerland
2
University of Sydney, School of Molecular Bioscience, Sydney, Australia
3
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California, USA
The Signal Recognition Particle (SRP) ribonucleoprotein complex and its membraneassociated receptor (SR) mediate co-translational targeting of membrane and secretory
proteins in all organisms. Ffh, the bacterial SRP54 homolog, recognizes the signal sequence
emerging from the translating ribosome, and the SRP RNA component serves as a platform
for the association of Ffh and the SR (FtsY in bacteria). The GTP binding domains of Ffh and
FtsY dimerize upon GTP binding. The complex then undergoes a large-scale rearrangement
from the initial position at the tetraloop to the distal region of the SRP RNA hairpin where
GTP hydrolysis is triggered and the complex dissociates. This allows the translating ribosome
to be delivered to the translocon in a GTP-dependent manner. The crystal structure of the
bacterial SRP:SR complex arrested in the cargo release state reveals that the Ffh:FtsY
heterodimer bind the 5’,3’ end of the SRP hairpin RNA1. The high resolution crystal structure
of Ffh:FtsY heterodimers bound to SRP RNA at the tetraloop and the distal region allowed a
deeper understanding of the system2. The tetraloop interactions reveal the structural basis of
receptor recruitment and rationalize previously accumulated biochemical data. The distal
interactions, in combination with mutagenesis and biochemical experiments reveal the role of
the SRP RNA in stimulation of GTP hydrolysis. This mechanism couples GTP hydrolysis to
conformational changes necessary for signal sequence handoff to the translocon1,2.
References
[4] Ataide S.F., Schmitz N., Shen K., Ke A., Shan S.O., Doudna J.A., Ban N. (2011) “The crystal structure of the signal
recognition particle in complex with its receptor.” Science 331(6019): 881-886.
[5] Voigts-Hoffmann F., Schmitz N., Shen K., Shan S.O., Ataide S.F., Ban N. (2013) “The Structural Basis of FtsY
Recruitment and GTPase Activation by SRP RNA”. Mol.Cell 52. Epub 2013 Nov 6.
10
Exploring the conformational flexibility of human IgE with a
inhibitory Fab fragment
Nyssa Drinkwater1,2, Ben Cossins3, Anthony Keeble2, Andrew Beavil2, James McDonnell2,
Alistair Henry3 and Brian Sutton2
1
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of
Medicine, Monash University, 3800, Australia.
2
Randall Division of Cell & Molecular Biophysics and MRC & Asthma UK Centre in Allergic
Mechanisms of Asthma, King’s College London SE1 1UL United Kingdom
3
UCB-Celltech, SL1 3WE United Kingdom
E-mail: [email protected] and [email protected]* (*from April 7 2014)
Immunoglobulin E (IgE) binding to its receptor FcεRI on mast cells mediates the allergic
response, and the interaction is a validated target for therapeutic intervention in allergic
diseases such as asthma1,2. IgE is known to display an acutely and asymmetrically bent
conformation in the receptor-binding Fc region (a), which becomes even more acute upon
receptor engagement3. Presented here is the crystal structure of a complex formed between
IgE-Fc and two symmetrically bound Fab fragments of an anti-IgE antibody (b). The
structure shows that IgE-Fc can adopt a completely extended conformation (c), which
requires a drastic “unbending” of 120° from the previously characterised bent structure.
Molecular dynamics simulation reveals metastable conformations for free extended IgE-Fc,
together with a number of intermediate conformations along a pathway between the bent and
extended structures. These simulations together with ITC, FRET, and SPR analyses suggest
that the capacity to unbend is an intrinsic property of IgE-Fc and not induced by Fab binding.
Understanding the full range of conformations accessible to the free IgE molecule is key to
developing IgE-targeted therapies for allergic disease, and the research presented here has
been instrumental in the development of ‘antibody enabled small molecule drug discovery’ a
new method for the discovery of allosteric inhibitors of complex therapeutic targets.
References
[6] Gould, H.J. & Sutton, B.J. (2008) “IgE in allergy and asthma today”, Nature Reviews Immunology, 8, 205-17.
[7] Holgate, S.T., Djukanovic, R., Casale, T. & Bousquet, J. (2005) “Anti-immunoglobulin E treatment with omalizumab
in allergic diseases: an update on anti-inflammatory activity and clinical efficacy” Clinical and Experimental Allergy,
35, 408-16).
[8] Holdom, M.D., Davies, A.M., Nettleship, J.E., Bagby, S.C., Dhaliwal, B., Girardi, E., Hunt, J., Gould, H.J., Beavil,
A.J., McDonnell, J.M., Owens, R.J. and Sutton, B.J. (2011) “Conformational changes in IgE contribute to its uniquely
slow dissociation rate from receptor FcεRI” Nature Structural and Molecular Biology, 18, 571-6.
11
Insights into the recruitment of the PAN2-PAN3 deadenylase
complex to miRNA targets by the GW182/TNRC6 proteins
Mary Christie1, Andreas Boland1, Eric Huntzinger1, Oliver Weichenrieder1 and Elisa
Izaurralde,1
Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35,
72076 Tübingen, Germany
The PAN2-PAN3 deadenylase complex functions in general and miRNA-mediated mRNA
degradation and is specifically recruited to miRNA targets by the GW182/ TNRC6 proteins.
PAN3 is an adaptor protein that recruits the PAN2 deadenylase to mRNA targets. PAN3
contains three prominent regions: an N-terminal region that is predicted to be unstructured, a
central pseudokinase (PK) domain and a highly conserved C-terminal domain (C-term) that is
unique to the PAN3 protein family. We have determined the crystal structure of the PAN3
adaptor protein that, unexpectedly, forms intertwined and asymmetric homodimers.
Dimerization is mediated by a coiled coil that links an N-terminal pseudokinase to a Cterminal knob domain. Despite containing nonconservative substitutions in all the sequence
motifs required for kinase activity, the PAN3 pseudokinase domain is capable of binding
ATP and nucleotide binding is required for mRNA degradation in vivo. Through a combined
mutational and functional analysis, we have identified critical residues that mediate PAN3
interaction with PAN2 and TNRC6 proteins, as well as additional residues required for
mRNA degradation in vivo. The most remarkable structural feature is the presence of a
tryptophan-binding pocket at the dimer interface, which mediates binding to TNRC6C in
human cells. Collectively, our data reveal the structural basis for the interaction of PAN3
with PAN2 and the recruitment of the PAN2-PAN3 complex to miRNA targets by TNRC6
proteins [1].
References
[1] Christie M, Boland A, Huntzinger E, Weichenrieder O, Izaurralde E (2013) “Structure of the
PAN3 Pseudokinase Reveals the Basis for Interactions with the PAN2 Deadenylase and the GW182
Proteins”, Molecular Cell, 51:360-363
12
Structural studies of the cavin proteins provide new insights into
the mechanisms of caveolae formation
Oleksiy Kovtun1, Vikas Tillu1, WooRam Jung1, Natalya Leneva1, Nick Ariotti1, Kirill
Alexandrov1, Rob Parton1,2 and Brett Collins1
1
Institute for Molecular Bioscience, UQ, Brisbane, Australia
Centre of Microscopy and Microanalysis, UQ, Brisbane, Australia
2
Caveolea are bulb-shaped membrane invaginations abundant in many cell types, including
adipocytes, muscle fibers and endothelial cells. These membrane structures have distinct
roles in lipid regulation, intracellular trafficking, signaling and mechanosensation [1].
Caveola deficiencies cause developmental and postnatal system pathologies in heart, adipose
and muscle tissues in human patients and animal models. Many tumor types demonstrate
changed expression patterns for caveolar proteins.
Formation and stabilization of caveolae depends on the membrane-embedded adapter protein
caveolin and coat proteins called cavins [2]. The cavins are a unique protein family with 4
members in the human genome (cavin1-4) that share no sequence homology with known
proteins, and are therefore likely to utilize an unknown assembly mechanism to drive
multimerization and subsequent membrane sculpting.
Using interaction analysis of systematically truncated cavins we discovered a universal
domain that supports homo- and hetero-oligomerisation of these proteins. X-ray crystal
structure determination of the oligomerization domain revealed a homo-trimer folded in a
long continuous parallel coiled-coil structure. A striking feature of this structure is the
presence of a conserved surface patch enriched in positively charged basic amino acids.
Lipid-interaction and point mutagenesis assays have shown that the part of this cationic
surface forms lipid binding site with broad specificity to phosphatidylinositol (PI)
headgroups. It is not clear however whether this PI-binging site targets cavins to the plasma
membrane or maintains caveolar pool of PIP2.
Electron microscopy of the full-length cavins reveals elongated bar-shaped structures that
resemble the surface structures of caveolae observed in vivo. Thus we propose that a
superhelical region represents the main structural domain of the cavin coat, is required for
assembly of higher-order cavin assemblies, and mediates binding to membranes and
membrane curvature essential for caveola formation.
References
[1] Parton RG, de Pozo MA (2013) “Caveolae as plasma membrane sensors, protectors and organizers”, Nat Rev Mol Cell Biol, 2:98-‐112. [2] Ariotti N, Parton RG (2013) “SnapShot: Caveolae, Caveolins, and Cavins”, Cell, 154:704 e1 CRYSTAL29 – 29th Conference of SCANZ - 2014
13
SESSION 6: CRYSTAL ENGINEERING
Chair: Stuart Batten, Monash
Exploiting redox-activity in metal-organic frameworks and porous
coordination polymers for functional properties
INVITED SPEAKER
Deanna M. D’Alessandro,1 Thomas B. Faust,1 Carol Hua,1 Chanel Leong,1 Weibin Liang,1
and Pavel M. Usov1
1
School of Chemistry, The University of Sydney, New South Wales 2006, Australia.
The development of redox-active and conducting microporous materials are highly sought
after goals: at a fundamental level these materials offer unprecedented insights into electron
delocalisation in three-dimensional coordination space; at an applied level, they have
potential for electrocatalytic conversion through to solar energy harvesting.1
This presentation will detail our latest results in the design and synthesis of Metal-Organic
Frameworks and Porous Coordination Polymers that integrate molecular components for
electron transfer including radical ligands, metal centres and mixed-valence clusters. The
application of solid-state DC and AC electrochemical methods, as well as the development of
solid-state near-IR/Vis and EPR spectroelectrochemical techniques will be described which
provide powerful in situ probes for the electron transfer characteristics of redox-active
materials.2 Our initial work on the theoretical and computational modelling of the electronic
and optical properties of these systems will also be detailed.3-5 The outcomes of this research
on both a fundamental and applied level pave the way towards advanced multifunctional
materials.
References
[1] D’Alessandro D.M., Kanga J.R.R. and Caddy J.S. (2011) “Towards conducting metal-organic frameworks”, Australian
Journal of Chemistry, 64:718-722.
[2] Usov P.M., Fabian C. and D'Alessandro D.M. (2012) “Rapid determination of the optical and redox properties of a
metal-organic framework via in situ solid state spectroelectrochemistry”, Chemical Communications, 48:3945-3947.
[3] Leong C.F., Faust T.B., Turner P., Usov P.M., Kepert C.J., Babarao R., Thornton A.W. and D’Alessandro D.M. (2013)
“Enhancing selective CO2 adsorption via chemical reduction of a redox-active metal-organic framework”, Dalton
Transactions, 42: 9831-9839.
[4] Liang W., Babarao R. and D’Alessandro D.M. (2013) “Microwave-assisted solvothermal synthesis and optical
properties of tagged MIL-140A metal-organic frameworks”, Inorganic Chemistry, 52:12878-12880.
[5] Leong C.F., Chan B., Faust T.B., Turner P. and D’Alessandro D.M. (2013) “Electronic, optical, and computational
studies of a redox-active napthalenediimide-based coordination polymer”, Inorganic Chemistry, 52:14246-14252.
14
Flexible crystals: Stretching the boundaries of a single crystal
Jack K. Clegg1, John C. McMurtrie,2 Anna Worthy, 2 Michael Pfrunder1 and Aidan Brock1
INVITED SPEAKER
1
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane St Lucia,
QLD, Australia, 4072
2
School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology,
Brisbane 4001, Australia
A crystal is normally thought of as a homogenous solid formed by a periodically repeating,
three-dimensional pattern of atoms, ions, or molecules. Indeed, the regular arrangement of
molecules, in a single crystal lead to many useful characteristics (in addition to diffraction!)
including unique optical and electrical properties, however, molecular crystals are not
typically mechanically robust, particularly compared to crystals of network solids like
diamond. Upon the application of stress or strain, these crystals generally irreversibly deform,
crack or break resulting in the loss of single crystallinity.
We have recently discovered a class of crystalline compounds that display the intriguing
property of elastic flexibility – that is they are capable of reversibly bending without
deforming, cracking or losing crystallinity. A number of these crystals are flexible enough to
be bent into a loop! (See Figure 1). We hypothesise that these intriguing properties stem from
the nature and arrangement of intermolecular forces present between molecules in the crystal
lattice, whereby weak interactions allow molecules to move sufficiently within a crystal
lattice (resulting in flexibility) while stronger interactions between molecules maintain the
crystallinity. This in turn leads to a loss of periodicity due to irregular compressions and
expansions of intermolecular distances throughout the lattice, challenging the definition of a
crystal. When the force is removed the crystals return to their perfectly ordered state. We are
currently investigating ways that we can tune the physical and mechanical properties of
materials through the combination and control of intermolecular interactions.
Figure 1: A crystal of [Cu(acacBr)2] showing elastic flexibility
15
The presence of mixed valence rare earths inRMn2X2compounds
Shane Kennedy1, Jianli Wang1,2, Stewart Campbell3, Michael Hofmann4 and Shixue Dou2
1
The Bragg Institute, Australian Nuclear Science and Technology Organization, Lucas Heights, NSW,
Australia
2
Institute for Superconductivity and Electronic Materials, University of Wollongong, Wollongong,
NSW, Australia
3
School of Physical, Environmental & Mathematical Sciences, University of New South Wales,
Canberra, ACT, Australia
4
Forschungs-Neutronenquelle Heinz Maier-Leibnitz, Technische Universität München, Garching,
Germany
The RMn2X2 series (where R is a rare earth and X is Si or Ge) are layered intermetallic
compounds in which the R and Mn atoms lie in alternate layers, separated by layers of X
atoms. These compounds display a fascinating array of magnetic phases, due to the strong
dependence of the Mn–Mn magnetic exchange interaction on the intralayer near neighbor
distance, and the interplay between the magnetism of the 3d(Mn)and 4f(R) layers. Magnetic
phase transitions in these compounds are often accompanied by structural changes (strictions)
due to the strong magnetoelastic coupling of both R and Mn sites. Up to eight different
magnetic structures are observed for Mn–Mn near neighbor distances in the range from ~2.84
Å to ~2.92 Å, and it is possible to vary that distance by changing chemical composition or
temperature and by applying mechanical pressure or a magnetic field.
The crystalline lattice of members of this family is generally consistent with rare earth
valence of 3+. However compounds with Ce, Eu or Yb at the R site do not always follow the
trend of the rest of the series. Reports on these anomalous compounds generally ascribe the
departure to intermediate valence states of the rare earth ions. However our studies of a range
of pseudo-ternaries (such as PrMn2Ge2-xSix [1] and Pr1-yYyMn2Ge2 [2]) lead us to consider
whether mixed valence states may also occur, due to chemical strain distributions originating
in the mixed layer planes.
Here we report our diffraction (synchrotron X-ray and neutron) of these compounds, where
we find that some of these anomalous compounds are two-phase in nature suggesting that
mixed valence states do exist. These results highlight the inter-relation between magnetic
correlations and rare earth valence state via magneto-elastic coupling.
References
[1] Wang J.L., Kennedy S.J., Campbell S.J., Hofmann M. and Dou S.X. (2013), “Phase gap in
pseudoternaryR1−yRyMn2X2−xXxcompounds “, Phys. Rev. B 87:104401
[2] Wang J.L., Caron L., Campbell S.J., Kennedy S.J., Hofmann M., Cheng Z.X., Din M.F.Md., Studer A.J., Bruck E. and
Dou S.X., (2013), “Driving magnetostructuraltransitions in layered intermetallic compounds”, Phys. Rev. Letters,
110:217211
16
Halogen-bond mediated crystal engineering of metal complexes
Michael Pfrunder1, Llew Rintoul1, Dennis Arnold1, Madeleine Shultz1, Jack Clegg2 and John
McMurtrie1
1
School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology,
Brisbane, 4001, Australia.
2
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia
Email: [email protected]
There is considerable interest in the synthesis of solid state crystalline materials with predictable
structural parameters and tunable physical properties. Transition metal complexes display a wide
variety of stereochemistries and exhibit diverse optical and magnetic properties, making them
attractive molecular components for this purpose.[1] Intermolecular interactions can be used to
influence the self-assembly of molecular components leading to predictable crystalline
supramolecular topologies.[2]-[4]
Halogen bonds (X-bonds) are electrostatic interactions between the partially positive site of a
polarized halogen (X-bond donor Cl, Br, I) and an electron rich species such as a Lewis base
(X-bond acceptor N, O, X-). X-bonds have been shown to be reliable interactions for the
production of predictable supramolecular structures with organic molecular
components.[5]We are investigating the potential to exploit X-bonding for the predictable
assembly of supramolecular architectures involving metal complexes.
We have prepared a range of crystals in which cationic metal complexes are encapsulated in
X- bond frameworks comprised of a range ofiodofluorobenzeneX-bond donors and halide Xbond acceptors. Synthesis, common packing motifs and design principles for these
supramolecular systemswill be discussed along with the impacts on spin-crossover behaviour
of complexes as a result of encapsulation in X-bond frameworks.
Halogen-bond
framework
Encapsulated metal
complexes
References
[1]
[2]
[3]
[4]
[5]
Sauvage J. (1999), Transition Metals in Supramolecular Chemistry, J Wiley & Sons, Baffin Lane.
Bertani R., Resnati G., Pilati T., Metranolo P., Terraneo G. et al. (2010) Coord. Chem. Rev. 254: 677-695.
Brammer L., Espallargas M.E. and Libri S. (2008) CrystEngComm., 10:1712-1727 .
Metrangolo P., Meyer F., Pilati T., Resnati, G. and Terraneo G. (2008) Angew. Chem. Int. Ed., 47:6114-6127.
Metrangolo P., Resnati G., Pilati T. and Biella S. (2008) Struct. Bonding (Berlin, Ger.), 126: 105-136.
17
Spin crossover induced molecular mosaic patterning generated in
a 3D Porous MOF
Natasha F. Sciortino,1 Suzanne M. Neville,1 Gregory J. Halder,2 Keith S. Murray,3 Boujemaa
Moubaraki,3 Jean-François Létard,4 Cameron J. Kepert1
1
School of Chemistry, University of Sydney,NSW 2006, Australia
X-ray Science Division, Advanced Photon Source, Argonne National Lab,, IL 60439, USA
3
School of Chemistry, Monash University, VIC 3800, Australia
4
Institut de Chimie de la Matie`re Condense´e de Bordeaux, UPR CNRS 9048, France
2
In the pursuit of smart high-performing materials, the recent integration of spin-crossover
(SCO) centers into porous framework materials gives rise to a unique molecular scenario in
which factors that govern the spin switching response (e.g., temperature, pressure, light,
magnetic field, and chemical environment) are newly intertwined with highly cooperative
structure–function relationships and dynamic host–guest chemistry.[1-3] With this design
approach, novel emergent capabilities arising from uniquely coexisting properties can be
harnessed to rationally design intelligent multifunctional materials that are purposefully
responsive to physical or physicochemical stimuli and targeted towards useful and
technological applications.
Here we present a porous bimetallic thermo- and photo-chromic SCO MOF that has spinstate switching centers embedded into an elastically frustrated scaffold, which generates a
unique emergent smart functionality due to the interplay between magnetic, electronic and
structural properties. By exploiting the inherent geometric frustration of a novel 3D Kagome
trihexagonal lattice, the onset of SCO induces a reversible volume quadrupling structural
modulation and a unique 3D long-range patterned array of distinct high-spin (HS) and lowspin (LS) domains (Figure 1a). This, through preorganization, results in an exceptional
complex, yet ordered nanopatterning effect (Figure 1b). Temperature-dependent powder and
single-crystal diffractometry using lab- and synchrotron-source X-rays reveals this SCO
behavior occurs due to the interplay between electronic spin-state switching with both
internal host-guest-pressure and geometric frustration effects. Furthermore, we discuss guestand light-induced properties.
Figure 1 - a) Structural representation of the MOF in its fully HS state (left) and after SCO
(right) highlighting the arrangement of HS and LS domains; and b) Schematic representation
of the Kagome lattice and the molecular mosaic nanopatterning effect. Unique domains
(defined by the 1D channel shape and the ordering of HS and LS Fe(II) topological vertices)
are shown as different colors.
References
[1] Halcrow M.A. (2013) Spin-crossover materials: properties and applications, John Wiley & Sons, Ltd.
[2] Gütlich, P. and Goodwin, H.A. (2004) “Spin crossover: a perspective”, Top. Curr. Chem. 233: 1-47.
[3] Murray, K.S. and Kepert, C.J. (2004) “Cooperativity in spin crossover systems. Memory, magnetism and
microporosity”, Top. Curr. Chem. 233:195-228
18
Combined neutron scattering and DFT modelling studies of
functional materials
Samuel G. Duyker1, Vanessa K. Peterson1, Gordon J. Kearley1, Anibal J. Ramirez-Cuesta2
and Cameron J. Kepert3
1
Bragg Institute, Australian Nuclear Science and Technology Organisation, Lucas Heights 2234,
Australia.
2
ISIS Facility, Rutherford Appleton Laboratory, Oxfordshire, UK.
3
School of Chemistry, The University of Sydney, NSW 2006, Australia.
Coordination framework materials are attracting increasing attention for their wide array of
potential applications and interesting properties. Our approach to understanding the
behaviour of these materials combines experimental work with density functional theory
(DFT) calculations in order to identify the specific features of the materials that give rise to
the properties of interest. Such properties include selective gas adsorption, negative thermal
expansion (NTE, contraction upon warming), and even the interplay between the two.
Our crystallographic studies of the LnCo(CN)6 (Ln = La-Lu, Y) series of cyanide frameworks
have revealed large, composition-dependent NTE behaviour.[1] The linear relationship
between the magnitude of NTE and the ionic radius of the Ln metal (Figure 1a) enables finetuning of the behaviour, which is key to potential applications in perfectly counterbalancing
the positive thermal expansion of other materials. A unique feature of the materials’
structures is the locally unstable trigonal prismatic LnN6 coordination geometry, which is
stabilised by the framework connectivity. A DFT calculation of the vibrational dynamics of
the structure, validated by inelastic neutron scattering experiments, reveals an unusual low
energy NTE-contributing vibrational mode, wherein the LnN6 unit twists about its axis, with
an energy of only ~35 cm-1 (Figure 1b). This finding contrasts with the rigid unit modes
(RUMs) that are prevalent in other systems. The discovery of the flexibility of this LnN6 unit
has implications for the strategic design of future materials with even larger NTE or other
pronounced mechanical behaviours.
Figure 1 a) The Ln-dependent NTE across the LnCo(CN)6 series and b) the low energy RUM
and non-RUM vibrational modes in LnCo(CN)6 that are responsible for the NTE behaviour.
References
[1] Duyker SG, Peterson VK, Kearley GJ, Ramirez-Cuesta AJ and Kepert CJ (2013) “Negative thermal expansion in
LnCo(CN)6 (Ln = La, Pr, Sm, Ho, Lu, Y): mechanisms and compositional trends”, Angew. Chem. Int. Ed., 52:52665270.
19
SESSION 7: BEYOND STRUCTURE 2
Chair: Suzanne Neville, University of Sydney
Let the powers combine: hybrid methods in structural biology.
Jane R Allison
INVITED SPEAKER
Centre for Theoretical Chemistry and Physics and Institute of Natural and Mathematical Sciences,
Massey University, Auckland 0632, New Zealand
The combination of experimental and computational approaches to biomolecular structure
determination is becoming ever more popular. Indeed, the generation of model structures
compatible with X-ray crystallography or NMR spectroscopy data relies on computational
modelling. In the case of X-ray crystallography, a single-structure interpretation is generally
appropriate. However solution-state experimental data, such as those obtained from NMR
spectroscopy, are averages over potentially broad ensembles of structures and significant (on
a molecular level) time-scales, so that it is inappropriate and, in some cases, impossible to
search for a single structure in keeping with all of the data. I will discuss the general problem
of inferring an ensemble based on average values, and outline various means of accounting
for averaging, including the advantages and limitations of each. I will show how a
sophisticated technique that encourages sampling of different conformations while
simultaneously taking averaging into account is able to cope with situations in which
standard techniques fail.
20
Reverse electrostatic complementarity based on the crystal
structure of the pure protease inhibitor E64c
Ming Wen Shi and Simon Grabowsky
School of Chemistry and Biochemistry, The University of Western Australia, 35 Stirling Highway,
Crawley, WA, Australia
Common strategies of drug design are to rationally modify the substituents of a known
biologically active molecule, i.e. the pharmacophore approach. By varying the substituents,
besides steric properties, one can change the electrostatic potential of the molecule and alter
the intermolecular interactions between the designed molecule and the target enzyme. The
idea of reverse electrostatic complementarity is similar, but here the electrostatic properties of
the enzyme pocket – even if unknown – can be simulated from just the knowledge of the
crystal structure of the pure biologically active molecule. Hence, we are able to visualize the
polarization and charge distribution of the pocket. [1] In future work, in order to achieve a
functional inhibitor, these pocket properties can be exploited.
E64c is a potent cysteine protease inhibitor. [2] Computational studies have been done in
order to understand its inhibitory mechanism. [3] As the crystal structure of E64c was not
available for those studies, the crystal structure of the related E64d was used as an alternative
for simulations. E64d has a terminal ethyl ester group instead of a carboxylic acid group (in
E64c), which can severely change the electrostatic potential, intermolecular interaction and
the geometry of the molecule in the crystal structure. However, the actual inhibitor prior to
the nucleophilic attack of the thiol group from the enzyme is E64c. Recently, we have solved
the crystal structure of E64c and used the new information for the reverse electrostatic
complementarity approach.
References
[1] Shi M.W., Sobolev A.N., Schirmeister T., Luger P., Mebs S., Dittrich B., Charles B., Turner M., Stewart S., Bak J.M.,
Jayatilaka D., Spackman M., Chen Y-S., Engels B. and Grabowsky S., Chem. Commun., to be submitted
[2] Otto H-H. and Schirmeister T. (1997) “Cysteine proteases and their inhibitors”, Chem. Rev., 97:133-172. ,
[3] Mladnovic M., Junold K., Fink R.F Thiel., W., Schirmeister T. and Engels B. (2008) “Atomistic insights into the
inhibition of cysteine protease: First QM/MM calculations clarifying the regiospecificity and the inhibition potency of
epoxide- and aziridine-based inhibitors”, J. Phys. Chem. B., 112: 5458-5469.
21
Crystal structures of ancestral amino acid binding proteins reveal
protein specialization can occur through conformational selection
Colin Jackson and Benjamin Clifton
INVITED SPEAKER
Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia.
Ancestral protein resurrection was used to create common ancestors of contemporary amino
acid binding proteins to investigate the molecular expansion of this protein family. All
proteins were produced in Escherichia coli at very high levels (~100 mg/L) and were
hyperthermostable. Unexpectedly, in addition to their primary binding affinities (glutamate,
glutamine, etc.), all ancestors displayed significant binding affinity for arginine. To determine
the molecular basis for this binding promiscuity, high resolution crystal structures (1.5
Å) have been solved of the ancestor of contemporary glutamine binding proteins in complex
with both arginine and glutamine. We observe significant differences in the binding modes of
the two amino acids, suggesting that the binding promiscuity of this ancestral form is reliant
on the structure being able to sample multiple conformations. In contrast, the contemporary
proteins display far less conformational plasticity. These observations are consistent with a
model whereby evolutionary specialisation occurs through a process of conformational
selection.
22
Crystal structures of genomic island components from Australian
A. Baumannii strains
Bhumika Shah1, Stephen Harrop2, Ian Paulsen1 and Bridget Mabbutt1
1
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
School of Physics, University of New South Wales, Sydney, Australia
2
Acinetobacter baumannii is a multi-drug resistant pathogen emerging as a major health threat
in critically ill patients across hospital settings worldwide. Comparative analysis of genomes
across Acinetobacter spp. indicates that large portions of its genetic material are not fixed.
Acquired through lateral transfer, gene clusters form genomic islands (GIs) as distinct regions
of novelty which appear to encode adaptive traits. These features are implicated in properties
such as virulence, pathogenicity and drug resistance.
As GI components tend to display highly novel sequences, their tertiary structure
determination and subsequent fold recognition can prove keys to annotation of function. I
was able to access some first Australian isolates of this gram-negative bacillus, including
community and clinical strains. From 125 proteins expressed through a structural genomics
high-throughput pipeline, two crystal structures have been solved.
The 2.65 Åcrystal structure of A. baumannii epimerase WbjB reveals an enzyme of the
extended short chain dehydrogenase/reductase (SDR) family in an unusual hexameric form,
with cofactor NADP bound. The GI from which the enzyme derives clearly encodes discrete
steps of lipopolysaccharide biosynthesis, so likely influencing virulence factors for its host
pathogen. A second crystal structure, SDR-WM99c (2.4 Å) also proved to be an SDR
enzyme, in this case of classical subtype. This structure shows homology to a group of
uncharacterized dehydrogenase/reductases across several microbial organisms.
The
recurrence of the SDR family within my target pool signifies that the highly diverse functions
of this enzyme group are ideal for adaptive genetic componentry of pathogens.
23
X-ray wavefunction refinement – introduction, examples, validation
Simon Grabowsky1, Magdalena Woińska1,2, Joanna M. Bąk1,2 and Dylan Jayatilaka1
INVITED SPEAKER
1
The University of Western Australia, School of Chemistry and Biochemistry, 35 Stirling Highway,
Crawley WA 6009, Australia
2
University Warsaw, Faculty of Chemistry, Pasteura 1, 02-093 Warsaw, Poland
Electron-density analysis performed after multipole modelling (MM) [1] is the primary
source of chemical information from an X-ray diffraction experiment beyond pure geometry.
But it is known that there are problems with the used basis functions, so that, in particular,
heavier atoms and polar bonds cannot always be described satisfactorily.[2] There are
promising efforts to improve MM.[3]
Here, we present an alternative way to improve the electron-density modelling that does not
need multipoles. We term this novel way “X-ray wavefunction refinement (XWR)”. XWR is
a protocol that defines how to combine existing techniques to arrive at a final electron-density
model that reconstructs the measured data. In the first step, the geometry is determined using
Hirshfeld atom refinement,[4] which is based on a stockholder partitioning of quantummechanical aspherical electron densities. In the second step, the same wavefunction is fitted
to the experimental data to reproduce the diffraction pattern and simultaneously minimise the
molecular energy.[5] The XWR protocol involves embedding the molecule into a field of
point charges and dipoles as well as termination strategies to avoid overfitting.[6]
Results from an X-ray wavefunction refinement are not restricted to the analysis of electron
density: the full reconstructed density matrix is available. Therefore, chemical problems can
be tackled with the optimum tools for any given question including, e.g., experimentally
derived bond orders, electron-pair localisation information, or energetics.
We will present first applications of this protocol for a selection of organic (amino acids,
tripeptides, hydrogen maleate salts, sulfur-containing protease inhibitors) and inorganic
(siloxanes, sulfur dioxide) compounds, for which we measured high-resolution lowtemperature X-ray diffraction data. We will show geometry improvements, anisotropic
displacement parameters for hydrogens, anharmonic motion parameters for sulfur and
chlorine atoms, and improved total electron-density distributions in comparison to results
from multipole modelling. Moreover, we will discuss the contribution of the experimental
data to the final constrained wavefunction (experimental exchange-correlation interaction
density) and demonstrate how the experimentally derived orbital-based descriptors assist in
solving fundamental chemical problems.
References
[1] Koritsanszky T. S. and Coppens P. (2001) “Chemical applications of X-ray charge-density analysis”, Chem. Rev.,
101:1583-1627.
[2] Engels B., Schmidt T. C., Gatti C., Schirmeister T. and Fink R. F. (2012) “Challenging problems in charge density
determination: polar bonds and influence of the environment”, Struct. Bond., 147:47-97.
[3] Koritsanszky T., Volkov A. and Chodkiewicz M. (2012) “New directions in pseudoatom-based x-ray charge density
analysis”, Struct. Bond., 147:1-26.
[4] Jayatilaka D. and Dittrich B. (2008) “X-ray structure refinement using aspherical atomic density functions obtained
from quantum-mechanical calculations”, Acta Cryst. A, 64:383-393.
[5] (a) Jayatilaka D. (1998) “Wave function for beryllium from X-ray diffraction data”, Phys. Rev. Lett., 80:798-801; (b)
Jayatilaka D. and Grimwood D. J. (2001) “Wavefunctions derived from experiment. I. Motivation and theory”, Acta
Cryst. A, 57:76-86.
[6] Grabowsky S., Luger P., Buschmann J., Schneider T., Schirmeister T., Sobolev A. N. and Jayatilaka D. (2012) “The
significance of ionic bonding in sulfur dioxide: bond orders from X-ray diffraction data”, Angew. Chem. Int. Ed.,
51:6776-6779.
24
SESSION 8: MATERIALS AND METALS
Chair: Alison Edwards, ANSTO
Using hydrogels to control crystallization: synthesizing next
generation implant materials
Kathryn M. McGrath
INVITED SPEAKER
MacDiarmid Institute for Advanced Materials and Nanotechnology, School of Chemical and Physical Sciences,
Victoria University of Wellington, Wellington 6140, New Zealand.
Biominerals are complex composite materials comprised of interpenetrating bioorganic/inorganic components. They are used for a range of roles within the organism including
structural integrity, protection, stability, allowance of feeding and navigation. Biominerals are
synthesized from aqueous solution under benign ambient reaction conditions. Generally the
mineral component traditionally would be thought of as being chemically and physically
quite boring. However, when partnered up with appropriate bioorganic molecules, and
collectively patterned into intricate three-dimensional structures these inorganic minerals are
transformed displaying for example optical or electronic properties not normally associated
with the pure mineral.
Like many biominerals the structure of bone is comprised of many different levels of
hierarchy, from the nanometre to in some cases the metre length scale, each differing in the
self-assembly, morphology and/or pattern/structure of the bioorganic and mineral
components. Reconstructing such a complex material synthetically is a considerable
challenge. However since the times of the Mayans other simpler biominerals, such as mollusk
shell (nacre) have been used to aid bone reparation or as straight implant materials.
Furthermore, because of its properties, including its interaction with light, the formation of
synthetic nacre is a highly sought after target.
In vivo the bioorganic molecules are synthesized and undergo self assembly in synchronicity
with the sequestering of the metal ions, controlled mineralisation and development of
hierarchical structure. Replicating these modes of formation synthetically is not possible
however by understanding the different elements of the biological mechanism it is possible to
develop different synthetic pathways allowing production of a similar final material. Many
approaches have therefore been utilised in an attempt to form synthetic nacre. Our approach
is to utilise carbohydrate- and protein-based hydrogels in combination with specific
polymeric additives to replicate the carbohydrate and protein self assembly and thereby the
chemical and physical environments for subsequent mineralization [1].
Using this approach we have replicated some of the nanometre and micrometre length scale
elements of nacre, see figure. By coupling this process with 3D printing stand alone
materials, the structure of which bears a resemblance to bone, can be formed, see figure.
However understanding the crucial factors for achieving controlled nucleation, growth,
morphology and multiple length scale hierarchical structure remains challenging.
1
Left: native nacre. Middle: synthetic nacre fabricated using a hyrdogel template. Right: 3D printed variant.
Reference
[1] Munro, N H, Green, D W, Dangerfield, A and McGrath, K M (2011) “Biomimetic mineralization of polymeric
scaffolds using a combined soaking and Kitano approach, Dalton Transactions, 40:9259-9268.
25
Probing electrochemistry and crystal structure simultaneously
Neeraj Sharma
INVITED SPEAKER
School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia.
Electrochemical energy storage devices in the form of batteries are ubiquitous in society,
used in everything from children’s toys to mobile electronic devices, providing portable
power solutions. There is a continuous drive for the improvement of batteries. A large
proportion of the function of batteries arises from the electrodes, and these are in turn
mediated by the atomic-scale perturbations or changes in the crystal structure during an
electrochemical process (e.g. battery use). Therefore, a method to both understand battery
function and improve their performance is to probe the crystal structure evolution in situ
while an electrochemical process is occurring inside a battery.
Our work has utilized the benefits of in situ neutron diffraction (e.g. sensitivity towards
lithium) to literally track the time-resolved evolution of lithium in cathode materials used in
rechargeable lithium-ion batteries (Figure 1). With this knowledge we have been able to
directly relate electrochemical properties such as capacity and differences in charge/discharge
to the content and distribution of lithium in the cathode crystal structure. In addition, the
ability to test smaller samples (e.g. in coin cells) with in situ X-ray diffraction has allowed us
to probe other batteries types, such as primary lithium and ambient temperature rechargeable
sodium-ion batteries.
In situ experiments that probe the time-resolved structural evolution during charge/discharge
provide unparalleled insight into how electrodes evolve. Using this information a
comprehensive atomic-scale picture of battery functionality can be created and permutations
can be made to the electrodes that optimize battery performance. This talk will showcase
some of our recent results on electrode structural evolution with respect to electrochemistry
in fully functioning batteries.
Figure 1. The (left) battery used for in situ neutron diffraction studies on Wombat, highintensity powder diffractometer at ANSTO. A (middle) selected reflection of the cathode
(dark red) evolving as a function of time, correlated to the charge and discharge process in
the battery. Snapshots (right) of the cathode crystal structure at various potentials during
charge. Lithium is shown in green with shading indicating lithium occupancy and MnO6
octahedra are purple. The discharged and charged state show only one lithium
crystallographic position while during charge there are two lithium positions.
26
First observation of intersite valence transitions involving 4d and
5d ions, as revealed by high-pressure x-ray and neutron diffraction
Chris D. Ling1, Zixin Huang1, Brendan J. Kennedy1 Wojciech Miiller1,2 and Max Avdeev2
1
School of Chemistry, The University of Sydney, Sydney 2006, Australia.
Bragg Institute, ANSTO, PMB 1, Menai 2234, Australia
2
Solid-state compounds are generally thought of as consisting of ions with well-defined
oxidation states. While ionic bonds always have some degree of covalent character, the ionic
approximation is usually sufficient to understand their “crystal chemistry” in conjunction
with concepts such as bond valence sum (BVS) and effective ionic radius (IR). IR predicts
that an atom will shrink as its oxidation state increases. This occurs gradually as electrons are
removed within a shell (e.g., IR(Ir3+) = 0.68, IR(Ir4+) = 0.625, IR(Ir5+) = 0.57 Å in 6-fold
coordination), but removing the last electron of a shell produces a more pronounced change
(e.g., IR(Bi3+) = 1.03, IR(Bi5+) = 0.76 Å). For a compound with a suitable combination of
cations, it should therefore be possible to effect a net reduction in volume by transferring an
electron from one to the other. A change in temperature and/or pressure could make such a
valence state transition favourable; however, in practice, such transitions are extremely rare.
Bond valence
BVS sum
(b)
c (Å)
c (Å)
a, b /1.7 (Å)
a, b /1.7 (Å)
Here, we will present and discuss our observation of such a pressure-induced charge transfer,
from Bi to Ir (or Ru) in the hexagonal perovskites Ba3+nBiM2+nO9+3n (n = 0,1; M = Ir, Ru)[1-3]
(a) 5.95
using high-pressure
synchrotron x-ray and neutron powder diffraction. They all show ~1%
(a)
5.95
first-order volume contractions at room temperature above
14.8 5 GPa, due to the large reduction
c --------in the IR of Bi 5.90
when the 6s shell is emptied
on> oxidation,
compared to the relatively
14.8
14.7
5.90
negligible effect of reduction on the IR ofc Ir--------or Ru.
> These are the first such transitions
involving 4d and5.85
5d compounds,
number of cases ever observed. Ab
a and double the total 14.7
< ----14.6
b /1.73 interactions through
initio calculations suggest
that
magnetic
very short (~2.6 Å) M–M bonds
5.85
a
[4]
< ----14.6
b /1.73of their electronic states.
contribute to the finely
5.80 balanced nature
14.5
5.80
5.5
5.5
(b) 5.0
BVS
14.5
Bi4+
+Ir 2Ir4+
Bi
5.0
4.5
→
Bi5+
+
2Ir3.5+
Ir
Bi
4.5
4.0
4.0
3.5
0
3.5
2
0
4
2
GPa
P /(GPa)
4
6
8
6
8
P (GPa)
(Left) Bond valence sums for Bi/Ir vs. pressure for Ba3BiIr2O9 from NPD data. (Right) Structure of
Ba3BiIr2O9; O are red, Ba are green, BiO6 octahedra are purple and IrO6 octahedra are gold.
References
[1] Miiller W, Avdeev M, Zhou Q, Kennedy BJ, Sharma N, Kutteh R, Kearley GJ, Schmid S, Knight KS, Blanchard PER,
Ling CD (2012) “Journal of the American Chemical Society”, 134: 3265-3270.
[2] Miiller W, Avdeev M, Zhou Q, Studer AJ, Kennedy BJ, Kearley GJ, Ling CD (2011) “Physical Review B”, 84:
220406(R).
[3] Miiller W, Dunstan MT, Huang Z, Mohamed Z, Kennedy BJ, Avdeev M, Ling CD (2013) “Inorganic Chemistry”, 52:
12461-12467.
[4] Huang Z, Auckett JE, Blanchard PER, Kennedy BJ, Miiller W, Zhou Q, Avdeev M, Johnson MR, Zbiri M, Garbarino
G, Marshall WG, Gu Q, Ling CD (2014) “Angewandte Chemie – International Edition” in press.
27
Critical applications of high absolute accuracy XAFS
measurements to low-energy inelastic electron scattering
School of Physics, University of Melbourne,Parkville VIC 3010, Australia
X-ray absorption fine structure (XAFS) analysis is one of the most common synchrotron
techniques for robust determinations of structural properties of a range of materials of interest
to chemical, biological, and physical sciences. We demonstrate how unprecedented accuracy
in recent XAFS measurements [1,2] unlocks new information about electronic properties of
materials, specifically the electron inelastic mean free path (IMFP) [3]. We find that the
sensitivity of XAFS to this fundamental physical property in the low-energy region [4]
enables a new measurement technique, capable of probing energies previously inaccessible
by other experimental arrangements [5].
We apply our developments to copper, and find that we can readily distinguish between
different IMFP calculations and theoretical electron scattering models. Further, our results
have inspired a resurgence of interest in the development of low-energy IMFP theory [6], and
we show how the interpretation of XAFS analysis has lead to improved understanding of the
fundamental physics of electron energy losses and bulk plasmon excitations in solid-state
materials [7]. These developments are readily applicable to any material for which highaccuracy XAFS data may be obtained, and provide crucial information for a range of
spectroscopies including AES, EPES, and LEED. This work is relevant to several of the
commissions of the IUCr including the commissions on XAFS, International Tables, and
Electron Crystallography.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
Glover J.L., Chantler C.T, Barnea Z., Rae N., Tran C.Q., Creagh D.C., Paterson D. and Dhal B.B. (2008)
“Measurements of the X-ray mass attenuation coefficient and imaginary component of the form factor of copper”,
Phys. Rev. A, 78:052902(1-13).
Chantler C.T., Tran C.Q., Barnea Z., Paterson D., Cookson D. and Balaic D.X. (2001) “Measurement of the X-ray
mass attenuation coefficient of copper using 8.85-20 keV synchrotron radiation”, Phys. Rev. A, 64:062506(1-15).
Bourke J.D. and Chantler C.T. (2010) “Measurements of electron inelastic mean free paths in materials”, Phys. Rev.
Lett., 104:206601(1-4).
Bourke J.D., Chantler C.T., and Witte C. (2006) “Finite difference method calculations of X-ray absorption fine
structure for copper”, Phys. Lett. A, 360:702-706.
Chantler C.T. and Bourke J.D. (2010) “X-ray spectroscopic measurement of photoelectron inelastic mean free paths
in molybdenum”, J. Phys. Chem. Lett., 1:2422-2427.
Nagy I. and Echenique P.M. (2012) “Mean free path of a suddenly created fast electron moving in a degenerate
electron gas”, Phys. Rev. B, 85:115131(1-4).
Bourke J.,D. and Chantler C.T. (2012) “electron energy loss spectra and overestimation of inelastic mean free paths
in many-pole models”, J. Phys. Chem. A, 116:3202-3205.
28
Investigation and control of periodic microphases in composites of
block copolymers and ionic liquids
Kevin Jack1, Thomas Bennett2, Kristofer Thurecht2,3 and Idriss Blakey2,3
1
Centre for Microscopy and Microanalysis, 2Australian Institute for Bioengineering and
Nanotechnology and 3Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD
4067.
Block copolymers consist of two, or more, chemically distinct polymer chains which are
covalently bonded. In the absence of specific interactions between the individual chains the
blocks will tend to microphase-separate into a range of 1-, 2- or 3-D periodic morphologies;
the nature of which depends on the degrees of polymerization of the individual blocks and
their relative incompatibilities [1]. Such materials have found a variety of applications, e.g. in
photolithography [2] and as templates for mesoporous materials [3]. In all of these
applications it is desirable to modulate or control the morphologies that are adopted by
varying the physical properties of the polymers or interfaces or by kinetic methods such as
annealing [2].
Very recently, we have begun a research program to investigate the use of ionic liquids as
selective solvents to control the microphase separation of di- and tri-block copolymers in
order to better understand the structure-property relationships in these materials and to tune
the phases of the polymers. The block-copolymers are polymerised by anionic methods,
which afford control over the block lengths and the distribution of molecular weight, while
the micophases adopted where characterised using high throughput measurements of smallangle X-ray scattering at the Australian Synchrotron. To illustrate this, the effects of a range
of ionic liquids on the phase diagrams of well characterized polystyrene-b-poly(methyl
methacrylate) block copolymers are presented in this work.
References
[1] Cochran, E. W., Garcia-Cervera, C. J., Fredrickson, G. H. (2006), Macromolecules 39:2449-2451.
[2] Keen, I., Cheng, H., Yu A., Jack, K., Younkin, T., Leeson, M., Whittaker, A.; Blakey, I. (2014), Macromolecules,
47:276-283
[3] Garcia, B. C., Kamperman, M.; Ulrich, R., Jain, A., Gruner, S. M., Wiesner, U. (2009), Chem. Mater. 21:5397-5405
29
SESSION 9: BRAGG MEDAL LECTURE
Chair: Peter Colman, WEHI
30
Chair: Jacqui Gulbis, WEHI
Targetting multidrug resistance in Neisseria: A structural approach
Alice Vrielink
University of Western Australia, Perth, Australia
31
SESSION 11: DRUG TARGETS AND INHIBITOR DEVLEOPMENT
Chair: Helen Blanchard, Griffith University
Structural diversity among autotransporter proteins
Begoña Heras1, Jason J Paxman1, Makrina Totsika2, Kate M Peters2 and Mark A Schembri2
INVITED SPEAKER
1
Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University,
Melbourne VIC 3086, Australia.
2
Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences,
The University of Queensland, Brisbane QLD 4072, Australia.
Bacterial antimicrobial resistance is increasing at an alarming pace with fears that we could
return to the pre-antibiotic era. There is an urgent need to increase our understanding of the
mechanisms underlying bacterial resistance to identify new targets for therapeutic
intervention. Our work focuses on a major group of proteins, namely autotransporter (AT)
proteins that are used by bacteria to establish highly persistent infectious diseases.
AT proteins are the largest group of outer membrane and secreted proteins in Gram-negative
bacteria [1-2]. These proteins promote the formation of aggregated communities and
biofilms, which are critical strategies bacteria use to resist the host immune response and
antibiotics. Furthermore, AT proteins are highly immunogenic and are integral components
of human vaccines [3-4]. Despite their central role in bacterial pathogenesis and potential for
vaccine development, the precise molecular mechanism of how AT proteins function is still
unknown.
Investigating the structural diversity of AT proteins is essential for understanding their
mechanism of action. We have recently elucidated the structure of Antigen 43 (Ag43), an AT
protein from uropathogenic E. coli (UPEC) that self associates forming bacterial aggregates
and biofilms [5]. Our studies have shown how Ag43's L-shaped structure drives the
formation of cell aggregates via a molecular Velcro-like handshake mechanism [6].
Furthermore, our recent studies on other AT proteins from UPEC show unexpected structural
diversity among this family of virulence proteins.
References
[1]
[2]
[3]
[4]
[5]
[6]
Benz I and Schmidt MA (2011) “Structures and functions of autotransporter proteins in microbial pathogens”, Int J
Med Microbiol 301, 461-468.
Kajava AV and Steven AC (2006) “The turn of the screw: variations of the abundant beta-solenoid motif in passenger
domains of Type V secretory proteins”, J Struct Biol 155, 306-315.
Serruto D, Bottomley MJ, Ram S, Giuliani MM and Rappuoli R. (2012) “The new multicomponent vaccine against
meningococcal serogroup B, 4CMenB: immunological, functional and structural characterization of the antigens”,
Vaccine 30 Suppl 2, B87-97.
Poolman JT and Hallander HO (2007) “Acellular pertussis vaccines and the role of pertactin and fimbriae”, Expert Rev
Vaccines 6, 47-56.
Klemm P, Hjerrild L, Gjermansen M and Schembri MA (2004) “Structure-function analysis of the self-recognizing
Antigen 43 autotransporter protein from Escherichia coli”, Mol Microbiol 51, 283-296.
Heras B, Totsika M, Peters KM, Paxman JJ, Gee CL, Jarrott RJ, Perugini MA, Whitten AE and Schembri MA (2013)
“The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping”,
Proc Natl Acad Sci U S A. 2013 Dec 13 [Epub ahead of print]
32
Crystal structure and immunological properties of the first annexin
from Schistosoma mansoni
Andreas Hofmann1,2, Chiuan Yee Leow3,4,5, Charlene Willis1,3, Asiah Osman1, Lyndel
Mason1, Anne Simon6, Brian J. Smith7, Robin B. Gasser2 and Malcolm K. Jones3,4
1
Structural Chemistry Program, Eskitis Institute, Griffith University, Brisbane, Queensland, Australia
Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia
3
Queensland Institute of Medical Research, Herston, Queensland, Australia
4
School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia
5
Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia
6
Université Lyon 1, Villeurbanne, and Laboratoire Chimie et Biologie des Membranes et des
Nanoobjets, Université Bordeaux, Pessac, France
7
La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
2
Schistosomiasis is a major parasitic disease of humans, second only to malaria in its global
impact. The disease is caused by digenean trematodes that infest the vasculature of their
human hosts. These flukes are limited externally by a body wall composed of a syncytial
epithelium, the apical membrane of which is a parasitism-adapted dual membrane complex.
Annexins are widespread throughout the Eukaryota, and members of this protein family have
become apparent in molecular studies of diverse parasite taxa, ranging from the diplomonad
protist Giardia to the neodermatan platyhelminths Taenia and Schistosoma (Hofmann et al.,
2010; Cantacessi et al., 2013). These proteins are thought to be of integral importance for the
stability of this apical membrane system.
We present the first structural and immunobiochemical characterisation of an annexin from
Schistosoma mansoni. The crystal structure of annexin B22 confirms the presence of the
previously predicted alpha-helical segment in the II/III linker (Hofmann et al., 2010) and
reveals a covalently linked head-to-head dimer. From the calcium-bound crystal structure of
this protein, canonical type II, type III and B-site positions are occupied, and a novel binding
site has been identified. The dimer arrangement observed in the crystal structure suggests the
presence of two prominent features, a potential non-canonical membrane binding site and a
potential binding groove opposite to the former (Leow et al., 2014).
Results from transcriptional profiling during development show that annexin B22 expression is
correlated with life stages of the parasite that possess the syncytial tegument layer, and ultrastructural
localisation by immuno-EM confirms the occurrence of annexins in the tegument of S. mansoni. Data
from membrane binding and aggregation assays indicate the presence of differential molecular
mechanisms and support the hypothesis of annexin B22 providing structural integrity in the tegument
(Leow et al., 2014).
References
[1] Hofmann A, Osman A, Leow CY, Driguez P, McManus DP and Jones MK (2010) “Parasite annexins – new molecules
with potential for drug and vaccine development”, BioEssays, 32:967-976.
[2] Cantacessi C, Seddon JM, Miller TM, Lee CY, Thomas L, Mason L, Willis C, Walker G, Loukas A, Gasser RB, Jones
[3]
MK and Hofmann A (2013) “A genome-wide analysis of annexins from parasitic organisms and their vectors”, Sci
Reports, 3:2893.
Leow CY, Willis C, Osman A, Mason L, Simon A, Smith BJ, Gasser RB, Jones MK and Hofmann A (2014) “Crystal
structure and immunological properties of the first annexin from Schistosoma mansoni - Insights into the structural
integrity of the schistosomal tegument”, FEBS J, in press.
33
The structure of a chorismate-utilizing enzyme from Mycobacterium
tuberculosis supports the possibility of developing a “magic shotgun” rather than a “magic bullet”
Genevieve Evans1, Ghader Bashiri1, Jodie M. Johnston1, Esther M.M. Bulloch1, Alexandra
Manos-Turvey2,3, Richard J. Payne2, Edward N. Baker1 and J. Shaun Lott1
1
Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for
Molecular Biodiscovery, The University of Auckland, Auckland 1010, New Zealand
2
School of Chemistry, Building F11, The University of Sydney, Camperdown, NSW 2006, Australia.
3
Present Address: Department of Biological Sciences, University of Pittsburgh, A321 Langley Hall,
4249 Fifth Avenue, Pittsburgh, PA 15260
Inhibiting the tryptophan biosynthesis pathway of Mycobacterium tuberculosis (Mtb) is lethal
to the bacterium due to synergy with the CD4 T-cell-mediated killing that is part of the host
immune response to infection.1 6-fluoro-anthranilate was recently shown to clear Mtb
infection in mice and human macrophages by appearing to function through inhibition of
anthranilate synthase (AS), the first step of the tryptophan biosynthetic pathway.1 We have
determined the first structure of Mtb-AS (Rv1609; trpE) refined to 2.1 Å resolution with a
crystallographic Rfactor=20.76 % and Rfree=24.17 %.
The Mtb-AS structure highlights the potential for a “magic shotgun” rather than a “magic
bullet” approach i.e. targeting multiple biosynthetic pathways with the same compound. MtbAS shares its structural fold and its substrate, chorismate, with Mtb salicylate synthase (SS).2
The latter catalyses the first committed step of another essential pathway for Mtb survival,
that of siderophore biosynthesis.3 The structure of Mtb-AS has an inhibitor, an isochorismate
mimetic which we initially developed against Mtb-SS,3 bound in the active site, and is the
first structure of an AS with an inhibitor bound.
The Mtb-AS structure shows that the inhibitor binds in a similar manner to that observed
previously for Mtb-SS (PDB ID 3VEH).4 The inhibitor has an IC50 value of 16.9 ± 0.8 µM
against Mtb-AS, which corresponds to a predicted Ki of the same value as that determined
against Mtb-SS.5 These data, along with the similar potency pattern of an inhibitor series
tested against Mtb-AS and Mtb-SS, indicate the possibility of designing an anti-TB drug
which disrupts both tryptophan and siderophore production in Mtb. Further compounds are
being pursued for their potential to inhibit both enzymes.
References
[1] Zhang Y.J., Reddy M.C., Ioerger T.R., Rothchild A.C., Dartois V., Schuster B.M. Trauner A., Wallis D., Galaviz S.,
Huttenhower C., Sacchettini J.C., Behar S.M. and Rubin E.J. (2013), “Tryptophan biosynthesis protects Mycobacteria
from CD4 T-cell-mediated killing”, Cell, 155:1296-1308.
[2] aHarrison A.J., Yu M., Gårdenborg T., Middleditch M., Ramsay R.J., Baker E.N., Lott J.S. (2006) “The structure of
MbtI from Mycobacterium tuberculosis, the first enzyme in the biosynthesis of the siderophore mycobactin, reveals it to
be a salicylate synthase, J Bacteriol, 188:6081-91.
b
Zwahlen J., Kolappan S., Zhou R., Kisker C., Tonge P.J. (2007) “Structure and mechanism of MbtI, the salicylate
synthase from Mycobacterium tuberculosis”, Biochemistry, 46:954-64.
[3] Chi, G., Manos-Turvey, A., O'Connor, P. D., Johnston, J. M., Evans, G. L., Baker, E. N., Payne, R. J., Lott, J. S.,
Bulloch, E. M. (2012) Implications of binding mode and active site flexibility for inhibitor potency against the
salicylate synthase from Mycobacterium tuberculosis, Biochemistry, 51:4868-79
[4] Manos-Turvey A., Bulloch E.M., Rutledge P.J., Baker E.N., Lott J.S. and Payne R.J. (2010) Inhibition studies of
Mycobacterium tuberculosis salicylate synthase (MbtI), ChemMedChem, 5:1067-79
34
Bacterial disarmament: DSB proteins as anti-virulence targets
Róisín M. McMahon1, Philip M. Ireland2, Kieran Rimmer3, Lakshmanane Premkumar1,
Mitali Sarkar-Tyson2, Craig Morton4, Martin J. Scanlon2, 4and Jennifer L. Martin1
1
Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072 Australia
Defence Science and Technology Laboratory, Porton Down, UK
3
Monash University, Department of Medicinal Chemistry, Parkville, Victoria, 3052 Australia
4
ARC Centre for Coherent Xray Science, Monash University, Parkville, Victoria, 3052, Australia
5
Biota Holdings Limited, Unit 10, 585 Blackburn Road, Notting Hill, Victoria 3168, Australia
2
A rapid rise in antibiotic resistance coupled with a decline in new antibiotics is generating a
public health crisis of global significance; new therapeutic solutions for bacterial infections
are urgently sought. For the next generation of drugs, it is hypothesized that a strategy of
targeting bacterial virulence may be preferable to that of targeting bacterial growth. Antivirulence agents that disarm but do not kill bacteria are anticipated to have a reduced
propensity to induce resistance, offering the potential for effective drugs with a longer
clinical lifespan.
DiSulfide Bond (DSB) proteins, a family of disulfide oxidoreductases that catalyse oxidative
folding and play an essential role in the assembly of many bacterial virulence factors are thus
a highly promising target for inhibitor development. Knock out of DiSulfide Bond protein A
(DsbA) has pleotropic effects upon virulence both in vitro and in in vivo models of infection
in a number of clinically relevant bacteria including Pseudomonas aeruginosa1and
Burkholderiapseudomallei2.
Our aim is to develop DSB inhibitors by combining structural characterization of a library of
bacterial DSB proteins with fragment based drug discovery programs. In this vein, we have
recently determined the high-resolution crystal structure of B. pseudomallei DsbA2, and also
conducted a fragment library screen against P. Aeruginosa DsbA (PaDsbA) that identified
small molecules that bind selectively to PaDsbA on the posterior face of the protein. For the
tightest binding fragment, NMR and X-ray structures provide high-resolution information
about the molecular nature of fragment binding and a starting point for hit-to-lead
elaboration.
We have also conducted comparative structural analyses of a library of 13 well-characterized
DsbAproteins to explore the druggability of this family of enzymes, identifying two distinct
structural classes (classes I and II)3. Class I and II are differentiated by their central β-sheet
arrangements and roughly separate the Gram-negative from Gram-positive DsbAs. These
classes can be further subdivided into a total of four subclasses on the basis of surface
features. In the context of inhibitor development, thesediscrete structural subclasses provide a
platform for developing DsbA inhibitors with subclass-wide spectrum of activity.
References
[1]
[2]
[3]
Urban A., Leipelt M., Eggert T. and Jaeger K.E. (2001), “DsbA and DsbC affect extracellular enzyme formation in
Pseudomonas aeruginosa”, J. Bacteriol. 183:587-596
Ireland P.M., McMahon R.M., Marshall L., Halili M., Furlong E., Tay S., Martin J.L., Sarkar-Tyson (2013),
“Disarming Burkholderiapseudomallei: structural and functional characterization of a disulfideoxidoreductase (DsbA)
required for virulence in vivo”, Antioxid Redox Signal: Sep 20 Epub. doi: 10.1089/ars.2013.5375. (Co-first author)
McMahon R.M., Lakshmanane P. and Martin J.L. (2013), “Four structural subclasses of the antivirulence drug target
disulfide oxidoreductase DsbA provides a platform for design of subclass-specific inhibitors. (Co-first author)
Revised version under review for a special issue of BBA: Proteins& Proteomics.
35
Design, development and validation of galectin-3-specific inhibitors
Khuchtumur Bum-Erdene and Helen Blanchard
Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, 4222 Australia.
Email: [email protected] and [email protected]
Galectin-3 is a β-galactoside-binding protein that is involved in inflammation and cancer
progression. In cancer, galectin-3 has been shown to induce proliferation of cancer cells,
metastasis and increased resistance to apoptosis inducing treatments. The design of selective
and potent inhibitors of human galectin-3 is necessary for use as potential therapeutics or as
molecular probes. Traditionally, inhibitor design has been based on a galactose framework, as
it is the natural substrate. Previous work from our group and others showed that saccharidebased small molecule inhibitors of galectins are effective against cancer progression.
However, recent reports indicate that different galectins can play different roles in certain
cancers, as exemplified by galectin-4, which can suppress cancer progression and metastasis
in colon and pancreatic cancer models in stark contrast to galectin-3. As the leading
candidates for galectin-3 inhibitors, such as thiodigalactosides and modified citrus pectin are
largely non-selective, we have initiated the design of inhibitors based on an alternative talosebased framework that allows exploitation of additional interactions with the galectin-3
protein. Despite initial taloside-based inhibitors showing selectivity for galectin-4 and
galectin-8 over galectin-3, we have utilized in silico docking and modelling approaches to
design and optimize new galectin-3-specific, high affinity taloside based inhibitors that
exploit the unique features of galectin-3 near its binding pocket. The elucidation of the crystal
structure of galectin-3-inhibitor complexes confirms the binding mode predicted from in
silico docking. Preliminary results indicate that the inhibitors are capable of inducing
cytotoxicity in a variety of galectin-3-expressing cancer cells. Further work is being
performed to confirm the mode of action in cells.
36
Tetrahydrocarbazoles: a future broad-spectrum antibiotic?
Zhou Yin1, Louise R. Whittell1, Jennifer L. Beck1, Michael J. Kelso1 and Aaron J. Oakley1
INVITED SPEAKER
School of Chemistry, University of Wollongong, Wollongong, NSW, Australia.
The rise of so-called “superbugs” – antibiotic resistant infection-causing bacteria, is one of
the preeminent public health concerns of the 21st century. New classes of antibiotics with
novel modes of action are needed. We have used fragment screening to develop inhibitors of
Pol III β, an essential protein in bacterial DNA replication and repair. Pol III β is a
homodimeric protein forming rings that encircle double-stranded DNA. Binding to β is
essential to the function of several proteins in DNA replication and repair, which use a short
peptide “linear motif”, QL[S/D]LF to bind to a conserved pocket on β. The conservation of β
interactions with partners and its low cellular abundance (300-600 dimers per cell) make it an
attractive drug target.
Fragment screening by X-ray crystallography was used to identify a series of chemical
entities that bind to the peptide-binding pocket on β and inhibit its interaction. Expansion of
these hits identified a series of tetrahydrocarbazoles as a promising scaffold for lead
development, the best of which demonstrate low-micromolar binding affinity. Together with
a structural and thermodynamic dissection of linear motif interactions, we demonstrate a
strategy for inhibitor development that recapitulates the mechanism of peptide binding.
37
SESSION 12: CHEMICAL CRYSTALLOGRAPHY
Chair: Chris Howard, U Newcastle
Laue neutron diffraction: Applications in chemical crystallography.
David R. Turner1, Gregory S. Hall1, Adrian J. Emerson1, Laura J. McCormick1, Ross O.
Piltz2 and Alison J. Edwards2
INVITED SPEAKER
1
School of Chemistry, Monash University, Clayton, VIC 3800, Australia.
The Bragg Institute, Australian Nuclear Science and Technology Organization, Locked Bag 2001,
Kirrawee DC, NSW 2234, Australia.
2
Single crystal neutron diffraction is a highly informative technique for chemical
crystallographers, primarily owing to the lack of correlation between atomic number and
scattering power.[1] The absence of this relationship can allow elements with similar electron
density to be distinguished in many instances and also allows the nuclear position of light
elements, such as hydrogen, to be located to precision not possible using X-ray methods.
There are significant drawback of neutron methods, namely the scarcity of the equipment and
the low flux which necessitates long collection times and large sample sizes. Monochromatic
diffraction experiments typically require samples in the region of 3 mm3, not easily available
for many materials, and can require several days for data collection on cells of modest size.
The Laue method, utilizing a ‘white’ beam, allows smaller samples (~ 1 mm3) to be collected
in a shorter time scale (~ 1 day) allows neutron diffraction to be more accessible to chemical
crystallographers. Australia is uniquely placed for such research with the KOALA instrument
at the Bragg Institute, ANSTO.
Our own research utilizes neutron diffraction to precisely locate hydrogen atoms within
hydrogen-bonding networks or in unusual hydrogen-bonding interactions where X-ray data is
particularly unreliable. This talk will explore several case studies to demonstrate the
importance of neutron diffraction to our research including: (i) mixed planar/non-planar
amides in hydrogen-bonding heterocycles (figure, left),[2] (ii) ‘proton sharing’ between
oximates (figure, right) and (iii) the use and geometry of nitrile groups as hydrogen bond
acceptors.[3,4]
References
[1]
[2]
[3]
[4]
Edwards A.J. (2011), Aust. J. Chem., 64:869-872.
McCormick L.J., MacDonnell-Worth C.J., Platts J.A., Edwards A.J. and Turner D.R. (2013), Chem. Asian J., 8:26422651.
Emerson A.J., Edwards A.J., Batten S.R. and Turner D.R. (2014), CrystEngComm, 2014, in press. DOI:
10.1039/c3ce42031k.
Turner D.R., Edwards A.J. and Piltz R.O. (2012), CrystEngComm, 14:6447-6451.
38
Targeting multi-step spin crossover in flexible coordination
polymers
Natasha F. Sciortino,1 Florence Ragon,1 Boujemaa Moubaraki,2 Keith S. Murray,2 JeanFrançois Létard,3 Cameron J. Kepert1 and Suzanne M. Neville1
1
The School of Chemistry, The University of Sydney, Australia
The School of Chemistry, Monash University, Australia
3
Sciences Moleculaires, ICMCB, Universite Bordeaux 1, France
2
Spin crossover (SCO) complexes are of current interest because of their potential use in
advanced technologies, such as memory storage devices and molecular sensing. Many of
these applications require hysteretic and abrupt complete SCO transitions centred around or
near room temperature. Multi-step transitions, those that display three- or more regions of
thermally stable, distinct magnetic states are a frequent feature in the literature of late owing
to the enhanced possibilities these materials provide for additional information storage
capacity. We are interested in designing a rational platform for routinely achieving multistability and then exploiting this to systematically tune and optimize multi-stable SCO
properties and uncover emergent properties. Examples include a two-step SCO with a record
breaking intermediate plateau temperature region spanning 130 K (Figure (left)) and a
remarkable thermal-cycling-driven transformation from a one-step to two-step SCO (Figure
(right)).
39
Versatile nanoballs – multiple packings and multiple properties
Stuart R. Batten1
1
School of Chemistry, Monash University 3800, Australia
We have synthesised large (2.7 nm) spherical metallosupramolecules (‘nanoballs’) with
interesting properties [1-3]. Metal ions can be varied with retention of overall structure and
crystal packing. The molecular packing creates cavities within the solid state, and the crystals
will readily absorb solvents such as methanol, acetonitrile or acetone (which also changes the
magnetic properties), and also absorb significant amounts of hydrogen and CO2 (but not
CH4), pointing to a new class of porous materials. Other properties include switching
between two magnetic spin states (spin crossover) upon change in temperature or irradiation
of light, and size-selective catalysis. New packing arrangements of the nanoballs can then be
achieved through variation of the counteranions or nitrile solvent, leading to new phases with
different physical properties.
References
[1] Duriska M.B., Neville S.M., Moubaraki B., Cashion J.D., Halder G.J., Chapman K.W., Balde C. Létard J.-F., Murray
K.S., Kepert C.J. and Batten, S.R. (2009), Angew. Chem. Int. Ed., 48:2549.
[2] Duriska M.B., Neville S.M., Lu J., Iremonger S.S., Boas J.F., Kepert C.J. and Batten, S.R. (2009), Angew. Chem. Int.
Ed., 48:8919.
[3] Duriska M.B., Neville S.M., Moubaraki B., Murray K.S., Balde C. Létard J.-F., Kepert C.J. and Batten, S.R. (2012),
ChemPlusChem, 77:616.
40
Resolution, interpretation and modelling in chemical
crystallography – critical analysis – furthering chemistry!
Alison J. Edwards
Bragg Institute, Australian Nuclear Science and Technology Organization, Locked Bag 2001,
Kirrawee D.C., N.S.W. 2232
The 20th Century brought stunning advances in the field of Crystallography, commencing
from the investigation of the first X-ray diffraction patterns through to such tours-de-force as
the determination of the early structures of simple compounds, the structures of penicillin,
vitamin B12 and progressing to increasingly large and complex structures such as DNA,
proteins, the Ribosome and fundamental questions such as the understanding of quasicrystals.
In all of these cases, proper understanding of the materials brought the challenge of correctly
comprehending previously unknown structure elements or assemblies – and typically on the
basis of data lying at the limits of our various technical capabilities with sometimes more
than a dash of creativity in the analytical thinking.
Today we have a formidable technical arsenal at our disposal with which to address any
crystallographic problem we select, well-appointed laboratory instruments, major facilities
like synchrotrons and neutron sources and computing power which was inconceivable to the
early practitioners in our discipline. In churning out hundreds of structures a day it is perhaps
inevitable that the occasional error might reach the literature. Crystallography has been at the
forefront of the implementation of validation technology aimed at exerting some quality
control in the field and this has helped to reveal outright fraud in the publication of crystal
structures but this technology has perhaps led us into complacency. The employment of “inhouse” crystallographic referees armed with significant computing power but possessed of
minimal experience, knowledge or exposure to properly critical assessments of the work of
others has perhaps led us to a situation where no-one is accepting the responsibility for
critically assessing the crystallographic reports reaching the literature.
The ability to mis- or over-interpret data in Chemical Crystallography is being abetted by a
tendency to report “theoretically calculated structures” as either predicting or verifying the
results of Crystallographic studies when they may well do neither. The positions of light
atoms which are ill-determined in the presence of heavy atoms are asserted to be “found” in
locations where an absence of electron density would be truly remarkable and this assertion is
invalidly stated to have been “proved” by the combination of crystallography and theoretical
calculations. In some cases, such reports have adequate spectroscopic data to allow a critical
reader to assess the veracity of the claimed position, but in many instances the supplementary
data are woefully incomplete or inconsistent with the reported findings to the extent that one
might question whether “supporting information” is actually being checked against the
conclusions in the paper which it purports to support!
This talk will demonstrate the importance of low resolution neutron diffraction data and
critical model building in concert with high resolution X-ray diffraction data in proving the
location of hydrogen nuclei adjacent to heavy atoms and how by proper critical analysis such
findings can then be leveraged to other structures via spectroscopic understandings arising
from the neutron studies which is applicable to compounds for which neutron diffraction data
may never be available The importance of critical assessment of the refined structures
against the norms of physical and chemical reasonableness, to extract new understandings
and to ensure that alternative (less remarkable) interpretations are not ignored will be
discussed. The final question is – how should we reward those revealing demonstrable errors
in our literature? Valid criticism is fundamental to good science but there is little
encouragement to engage in it.
41
Local order in wüstite. Fe1-xO, using a PDF approach
T.R. Welberry, D.J. Goossens2 and A.P. Heerdegenr1
1
Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia.
Many minerals (and materials more widely) show evidence of strong and complex local
structural ordering. This local ordering can affect a material’s mechanical properties, its
transport properties (for example, how vacancies relate to oxygen transport through the
structure) and its thermodynamics, and so is clearly of prime importance. For crystalline
materials the analysis of single crystal diffuse scattering (SCDS) is the most definitive way of
determining local structure but for many minerals (and materials more widely) single crystals
of a sufficient size for such studies are often not readily obtainable and powder diffraction
data must suffice. While conventional powder XRD (e.g. using Rietveld refinement [1]) can
provide information about the average crystal structure, total scattering (TS) – which includes
both Bragg peaks and diffuse scattering – is needed if information about the local structure
and short-range order is to be gained. The pair distribution function (PDF) analysis of such
total scattering data has become a widely used technique for extracting such local structural
information from a wide variety of materials including crystalline powders, nano-materials,
amorphous materials, glasses and liquids [2–6].The aim of the present work is to explore the
sensitivity of the PDF methodology to various aspects of disorder and short-range order for a
mineral system for which the local structure has been characterised previously using SCDS.
The system chosen for this study is the non-stoichiometric iron oxide wüstite,(Fe1−xO, x =
0.057). The X-ray diffraction patterns obtained from a single crystal of wüstite show strong
and richly structured diffuse scattering (see Fig. 1).This has enabled a detailed model of the
defect structure to be established [7,8].The aim is to assess to what extent this defect structure
model could have been established using PDF analysis of powder diffraction data.
Fig.1 (a) Single crystal diffuse scattering. (b) Powder diffraction.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
Rietveld H.M. (1969), J. Appl. Cryst., 2:65.
Michel F.M., Ehm L., Antao S.M., Lee, P.L., Chupas P.J. and Parise J.B. (2007), “The structure of ferrihydrite, a
nanocrystalline material”, Science, 316(5832):1726–1729.
Juhas P., Cherba D.M., Duxbury P.M., Punch W.F. and Billinge S.J.L. (2006), “Abinitio determination of solid-state
nanostructure”, Nature, 440(7084):655–65.
Billinge S.J.L. and Levin I. (2007), “The problem with determining atomic structure at the nanoscale”, Science,
316(5824):561–565.
Proffen T.H. (2006), “Analysis of disordered materials using total scattering and the atomic pair distribution
function”, Neutron Scattering in Earth Sciences,63:255–274.
Huq A., Welberry T.R. and Bozin E. (2010), “Advances in structural studies of materials using scattering probes”,
MRS Bulletin, 35:520–530.
Welberry T.R. and Christy A.G. (1995), “A paracrystalline description of defect distributions in wustite, Fe1−xO”,
Journal of Solid State Chemistry, 117:398–406.
Welberry T.R. and Christy A.G. (1997), “Defect distribution and the diffuse X-ray Diffraction pattern of wustite,
Fe1−xO”, Physics and Chemistry of Minerals, 24:24–38.
42
What will the new Volume of the International Tables for
Crystallography be about? Volume I: XAS, Eds CT Chantler, B
Bunker, F Boscherini
Chris T. Chantler
School of Physics, University of Melbourne, Parkville, Victoria 3010, Australia
Research in core physics and atomic and condensed matter science are increasingly relevant
for diverse fields and are finding application in chemistry, engineering and biological
sciences, linking to experimental research at synchrotrons, reactors and specialized facilities.
A plethora of different approaches are popular in the literature and the Volume will hope to
capture their greatest achievements and value by representation of all the leading groups from
Europe, America, Asia and Australia (plus elsewhere!).
Over recent synchrotron experiments and publications methods have developed for
measuring the absorption coefficient far from the edge and in the XAFS (X-ray absorption
fine structure)region in neutral atoms, simple compounds and organometallics reaching
accuracies of below 0.02%. This is 50–500 times more accurate than earlier methods, and
50–250 times more accurate than claimed uncertainties in theoretical computations for these
systems. The data and methodology are useful for a wide range of applications, including
major synchrotron and laboratory techniques relating to fine structure, near- edge analysis
and standard crystallography. A comment on some key features of the new Volume in its
infancy are presented, and contributions, support and suggestions will be warmly welcomed
by all Editors.
References
[1]
[2]
[3]
[4]
Chantler C.T., Barnea Z., Tran C.Q., Rae N.A. and de Jonge M.D. (2012), “’Invited / Topical Review’ A step toward
standardization: Development of accurate measurements of X-ray absorption and fluorescence”, Journal of
Synchrotron Radiation 19: 851-862
Glover J.L. et al. (2008), Phys. Rev. A78:052902
Rae N.A. et al. (2010), Phys. Rev. A 81: 022904
Islam M.T. et al. (2010), Phys. Rev. A 81: 022903
43
SESSION 13: METHODS IN STRUCTURAL BIOLOGY
Chair: John Bruning, University of Adelaide
Fragment-based molecular replacement with Phaser
Airlie J. McCoy and Randy J. Read
INVITED SPEAKER
Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.
A “traditional” molecular replacement (MR) problem could be considered one where the data
extend to at least 2.5Å, where the template protein structure has more than 30% sequence
identity with the target and where the template represents at least a quarter of the scattering.
Phaser, in conjunction with suitable model editing procedures [1], generally succeeds in
solving these problems. This is now also the case in the presence of translational
noncrystallographic symmetry (tNCS), which, without tNCS-corrected likelihood functions,
could foil MR even with excellent templates [2]. However, MR problems are increasingly no
longer “traditional”, as researchers target proteins without good homologues and crystals of
protein complexes and assemblies for which only a small fraction of the scattering has a
template, but also can obtain data to higher resolution than they could previously due to
bright synchrotron sources and other technical advances, including Pilatus detectors.
Fragment based approaches to MR are hence a rising approach to structure solution. The
fragments may be small secondary structure fragments, single helices, or in the limit, single
atoms. A game-changing advance in this field has been the development of the expected LLG,
which predicts what the signal-to-noise in the MR search will be given the target, the
template and the data. Fragment based approaches to MR pose particular problems for
refinement and model building due to the very poor phases from the incomplete and/or
inaccurate initial substructure. Techniques hitherto associated with experimental phasing such
as density modification and direct methods can be used to improve map quality and pull
solutions from seemingly hopeless initial Phaser results [3,4].
References
[1] Bunkoczi G. and Read R.J. (2011) “Improvement of molecular replacement models with Sculptor”, Acta Cryst D,
67:303-312.
[2] Sliwiak J., Jaskolski M., Dauter Z., McCoy A.J. and Read R.J. (2014) “Likelihood-based molecular replacement
solution for a highly pathological crystal with tetartohedral twinning and sevenfold translational noncrystallographic
symmetry” Acta Cryst D, 70:471-480
[3] Rigden D.J., Keegan R.M. and Winn M.D. (2008) “Molecular replacement using ab initio polyaline models generated
with ROSETTA” Acta Cryst D, 64: 1288-1291
[4] Rodríguez Martínez D.D., Grosse C., Himmel S., González C., de Ilarduya I.M., Becker S., Sheldrick G.M. and Usón I.
(2009) “Crystallographic ab initio protein solution far below atomic resolution” Nature Methods 6:651-653
44
Structural insights into the role of the cyclic backbone in a squash
trypsin inhibitor
Norelle L. Daly1,3, Louise Thorstholm1, Kathryn P. Greenwood1, Gordon J. King1, K. Johan
Rosengren2, Begoña Heras1,4, Jennifer L. Martin1 and David J. Craik
INVITED SPEAKER
1
Institute for Molecular Bioscience
School of Biomedical Sciences, The University of Queensland Brisbane, 4072
3
Centre for Biodiscovery and Molecular Development of Therapeutics and School of Pharmacy and
Molecular Sciences, James Cook University, Cairns 4878
4
Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University,
Melbourne 3086
2
MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and on the
basis of its exceptional proteolytic stability is a valuable template for the design of novel drug
leads. Insights into inhibitor dynamics and interactions with biological targets are critical for
drug design studies, particularly for protease targets. Here we show that the cyclization and
active site loops of MCoTI-II are flexible in solution, but when bound to trypsin the active
site loop converges to a single well-defined conformation. This finding of reduced flexibility
on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which
suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a
possible explanation for this discrepancy based on degradation of the complex over time. Our
study also unexpectedly shows that the cyclization loop, not present in acyclic homologues,
facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with
trypsin.
45
Validation of ligands in X-ray crystal structures
Alpeshkumar K. Malde1, and Alan E. Mark1,2
1
School of Chemistry and Molecular Biosciences and 2Department for Molecular Bioscience,
University of Queensland, Brisbane, QLD 4072, Australia
As of early 2014, the Protein Data Bank (PDB) contained over 97,000 structures, of which
over 25,000 X-ray crystal structures contained at least one of >17,000 organic ligand
molecules. The number of ligand:protein structures in the PDB is growing rapidly
considering that over 9,000 structures were deposited in last five years. Current structure
refinement and validation protocols are well suited for biomolecular structures like proteins
and nucleic acids, but are far from optimal for non-covalently bound ligands in such
structures. The undetected errors and uncertainty in the ligand:protein structures can lead to
the misinterpretation of experimental data and possible failure of drug discovery efforts.
I will present case studies on the applications of molecular dynamics simulations and free
energy calculations in conjunction with well-validated ligand force field parameters in
validating and correcting the structure and binding pose(s) of ligands in X-ray crystal
structures.
References
[1] Koziara K.C., Stroet M., Malde A.K. and Mark A.E. (2014), “Testing and validation of the Automated Topology
Builder (ATB) version 2.0: Prediction of hydration free enthalpies”, Journal of Computer-Aided Molecular Design, in
press.
[2] Nair P.C., Malde A.K. Drinkwater N. and Mark A.E. (2012), “Missing Fragments: Detecting cooperative binding in
fragment based drug design”, ACS Medicinal Chemistry Letters, 3:322–326.
[3] Malde A.. and Mark A.E. (2011), “Challenges in determining the binding modes of non-standard ligands in X-ray
crystal complexes”, Journal of Computer-Aided Molecular Design, 25:1-12.
[4] Malde A.K., Zuo L., Breeze M., Stroet M., Poger D., Nair P.C., Oostenbrink C. and Mark A.E. (2011), “An Automated
force field Topology Builder (ATB) and repository: version 1.0.”, Journal of Chemical Theory and Computation,
7:4026–4037.
46
The structure of the megadalton magnesium chelatase assembly
Anthony P. Duff1, Shabnam T. Tabrizi2, Artur Sawicki2, Anna V. Sokolova1, Kathleen
Wood1, Tom Joss2, Alison M. Kriegel2, Robert D. Willows2,
1
2
Australian Nuclear Science and Technology organisation (ANSTO), Lucas Heights, NSW 2234
Macquarie University Department of Chemistry and Biomolecular Sciences, North Ryde, NSW 2109
We are using x-ray and neutron scattering to characterise the subunit structure of magnesium
chelatase, and crystallisation of isolated subunits is continuing. Magnesium chelatase
catalyses the insertion of Mg(II) into protoporphyrin IX to make magnesium protoporphyrin,
the first committed step in the biosynthesis of chlorophyll. The catalytic activity is driven by
AAA(+) ATPase subunit “I”. The core structure is composed of hexameric I and a related,
but non-ATPase hydrolysing hexameric “D”. Subunit I exists in oligomeric from or
hexameric form depending on nucleotide binding. The D subunit exists only as the hexamer.
Scattering results indicate that the hexameric ID core structure from Chlamydomonas
reinhardtii is significantly larger than expected, possibly a dynamic structure containing
additional loosely bound I subunits attached. The protoporphryn IX molecule is bound
jointly by a subunits H and GUN4. H and GUN4 are well characterised in isolation, but they
bind ambiguously to the core structure, as observed in SAXS and SANS experiments.
A more recently discovered highly homologous “I2” subunit substitutes for I subunits in the
complex. SANS data indicates that I2 has zero affinity for itself, but activity assays reveal a
dramatic modulation of the catalytic activity due to the additional presence of I2.
47
First structure of a non-processive pectin methylesterase (from
Aspergillus niger): comparison to processive orthologues from
plants and bacteria
Geoffrey B. Jameson1, Lisa M. Kent1, Gillian E. Norris1, Laurence D. Melton2, Davide
Mercadante3 and Martin A.K. Williams1
1
Institute of Fundamental Sciences, Massey University, Palmerson North, 4442, New Zealand
School of Chemical Sciences, University of Auckland, Auckland, New Zealand
3
Heidelberg Institute for Theoretical Studies, Schloß-Wolfsbrunnenweg, 69118 Heidelberg, Germany
2
Pectin methylesterases (PMEs) are deeply involved in plant cell wall modification through
hydrolysis of the homogalacturonan (HG) O6-methylester groups of pectin. PMEs are also
produced by bacteria and fungi to attack plant cell wells. Whereas bacterial and plant PMEs
are generally processive (de-methylesterification proceeds sequentially from one
galacturonide residue to the next), fungal PMEs are reportedly non-processive (demethylesterification occurs randomly). Through molecular dynamics simulations on
variously patterned decasaccharide homogalacturonan (HG) species we have probed the
mechanism of processivity1,2 in the structurally well-characterised3 bacterial PME from
Erwinia chrysanthemi (= Dickeya dadantii).
We report here the biochemical characterisation of the N-‐glycosylation 2-‐2
site (Asn84) closely homologous PME1 and PME2 from the fungal plant
1 3-‐2
pathogen Aspergillus niger, showing that relative to a orange
2 4-‐2
PME these enzymes are non-processive. The plant and
3
bacterial PMEs share ~30% sequence identity with each
other and with the fungal PMEs. The structures of Ani-PME2
4
Substrate-‐ 5
in completely deglycosylated (PNGaseF-treated) and Endo-H
binding 6
groove
treated forms (N-acetylglucosamine stub) have been
7
determined to better than 1.8 Å resolution (respectively,
8
9
Rwork (Rfree) 0.177 (0.208) and 0.184 (0.211)). The β helix
10-‐1
structure common to a several carbohydrate-processing
enzymes is also observed for PME2.
1: The 10⅔-‐turn parallel β The plant4,5 and especially the bacterial3 PMEs have Figure helix of the PME2 from A. niger. substantially longer loops surrounding the substrate-binding
site than the Ani-PME2, which differs further with a strongly negatively charged surface at
neutral pH, which may encourage release of the negatively charged product, rather than
relative movement of the PME along the homogalacturonan chain. Molecular dynamics
simulations are being conducted on PME2-HG complexes to understand better processivity
and non-processivity in PMEs.
References
[1] Mercadante M., Melton L.D., Jameson G.B., Williams M.A.K. and De Simone A. (2013) “Substrate dynamics in
[2]
[3]
[4]
[5]
enzyme action: rotations of monosaccharide subunits in the binding groove are essential for pectin methylesterase
processivity”, Biophysical Journal 104: 1731-1739.
Mercadante D., Melton L.D., Jameson G.B. and Williams M.A.K. (2013) “Processive pectin methylesterases: the role
of electrostatic potential, breathing motions and bond cleavage in the rectification of Brownian motions”, PLoS ONE 9:
e87581 (11 pages).
Fries M., Ihrig J., Brocklehurst K., Shevchik V.E. and Pickersgill R.W. (2007) “Molecular basis of the activity of the
phytopathogen pectin methylesterase", EMBO J 26: 3879-3887.
Di Matteo A., Giovane A., Raiola A., Camardella L., Bonivento D., De Lorenzo G., Cervone F., Bellincampi D,
Tsernoglou D (2005) “Structural basis for the interaction between pectin methylesterase and a specific inhibitor
protein”, Plant Cell 17: 849-858.
Johansson K., El-Ahmad M., Friemann R., Jörnvall H., Markovic O. and Eklund H. (2002) “Crystal structure of plant
pectin methylesterase”, FEBS Letters 514: 243-249.
48
SESSION 14: MEMBRANE PROTEINS
Chair: Simon Williams, University of Queensland
Structure, function, and inhibitors of the acid-gated Helicobacter
pylori urea channel, an essential component for acid survival
Hartmut Luecke
INVITED SPEAKER
Center for Biomembrane Systems, University of California, Irvine, CA 92697-3900, USA.
Half the world’s population is chronically infected with Helicobacter pylori, causing gastritis,
gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated
innermembrane urea channel, HpUreI, is essential for survival in the acidic environment of
the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid
access of urea to cytoplasmic urease. Urease produces NH3 and CO2, neutralizing entering
protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH
below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a
hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses
a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a
previously unobserved fold comprising a two-helix hairpin motif repeated three times around
the central axis of the channel, without the inverted repeat of mammalian-type urea
transporters. Both the channel and the protomer interface contain residues conserved in the
AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric
state in this superfamily. Predominantly aromatic or
aliphatic side chains line the entire channel and define
two consecutive constriction sites in the middle of the
channel. Mutation of Trp153 in the cytoplasmic
constriction site to Ala or Phe decreases the selectivity
for urea in comparison with thiourea, suggesting that
solute interaction with Trp153 contributes specificity.
The previously unobserved hexameric channel
structure described here provides a new model for the
permeation of urea and other small amide solutes in
prokaryotes and archaea. Follow-up microsecondscale unrestrained molecular dynamics studies now
provide a detailed mechanism of urea and water
transport by HpUreI.
References
[1] Strugatsky, D., McNulty, R.M., Munson, K., Chen, C.-K., Soltis, S.M., Sachs, G., Luecke H. “Structure of the protongated urea channel from the gastric pathogen Helicobacter pylori” Nature 493, 255–258 (2013).
[2] McNulty, R., Ulmschneider, J.P., Luecke, H., Ulmschneider, M.B. ”Mechanisms of molecular transport through the
urea channel of Helicobacter pylori” (2013) Nature Communications 4, article number 2900.
49
Structures of the full-length bitopic membrane protein CYP51 from
Saccharomyces cerevisiae provide insight into substrate and drug
binding and mutations affecting antifungal susceptibility
Joel D.A. Tyndall,1 Alia Sagatova,2 Thomas M. Tomasiak,3 Mikhail V. Keniya,2 Franzi U.
Huschmann,1,2 Joseph D. O’Connell III,3 Andrew Rodruigez,3 Janet Finer-Moore,3 Jeffrey G.
MacDonald,4 Richard D. Cannon,2 Robert M. Stroud3 and Brian C. Monk2
1
School of Pharmacy,University of Otago, Dunedin9054, New Zealand.
Sir John Walsh Research Institute and Department of Oral Sciences, Faculty of Dentistry, University
of Otago, Dunedin 9054, New Zealand.
3
Department of Biochemistry and Biophysics, University of California at San Francisco, San
Francisco, California 94158, USA.
4
Departmentof Molecular Genetics, University of Texas Southwestern Medical Center, Dallas,
TX75390, USA
2
Lanosterol 14α-demethylase (CYP51, Erg11p) is a CYP51 cytochrome P450 monooxygenase required for sterol biosynthesis and is the target of the azole antifungal drugs.
Elucidation of the complete structure of this enzyme is important for understanding how
substrates and inhibitors are recognized and bound. Resistance to commonly used azole drugs
can arise through mutations that reduce their binding affinity for the fungal enzyme. We
present high-resolution X-ray crystal structures of the full-length monospanning membrane
protein CYP51 from Saccharomyces cerevisiae(Erg11p) in both wild type and mutant forms,
with and without ligands bound to the catalytic heme. S. cerevisiaeERG11 was cloned with a
C-terminal 6×His tag at the PDR5 locus in S. cerevisiae ADΔ. The tagged CYP51 was
solubilised from membrane preparations with N-decyl-β-D-maltoside, purified by Ni-NTA
agarose affinity chromatography and Superdex 200 size exclusion chromatography, and its
structure determined by X-ray crystallography via molecular replacement using the soluble
domain of the human homologue. Structures were determined at resolutions ranging from 1.8
to 2.8 Å in the absence of added ligand, with the azole inhibitors itraconazole, fluconazole
and voriconazole or with the substrate lanosterol or the substrate mimicestriol bound within
the active site. The protein possesses two N-terminal helices oriented at ~60° to each other
that tether the enzyme to the membrane and orient the substrate channel relative to the lipid
bilayer. The structures revealed lanosterol bound within the active site, a substrate channel
linked to the lipid bilayer, and a proposed product exit channel. The intact enzyme displayed
less conformational heterogeneity between structures compared to many other N-terminally
truncated eukaryotic cytochrome P450s, suggesting that the transmembrane domain itself, or
associated hydrophobic molecules such as lipids or reaction product, may decrease
conformational heterogeneity. The S. cerevisiaeY140F and G73E mutations, which have
been reported to confer azole resistance in Candida albicans and Aspergillus fumigates
clinical isolates respectively, have been introduced and their corresponding structures
determined. The Y140F mutation showed normal binding of itraconazole to the mutated
enzyme. Cells containing this mutation became resistant to short-tailed, but not long-tailed,
azoles. These structures provide a practical basis for the design of therapeutics with the
potential to circumvent the development of resistance and minimize off-target effects.
50
Crystal structure determination of the integral membrane
diacylglycerol kinase
Dianfan Li1, Joseph A. Lyons1, Valerie E. Pye2, Lutz Vogeley1, David Aragão3, Colin P.
Kenyon4, Syed T. A. Sha1, Christine Doherty1,5, Margaret Aherne1 and Martin Caffrey1
1
School of Biochemistry and Immunology & School of Medicine, Trinity College Dublin, Dublin,
Ireland
2
Clare Hall Laboratories, Cancer Research UK, Blanche Lane, South Mimms, Hertfordshire, EN6
3LD, UK
3
Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria, VIC 3168, Australia
4
CSIR, Biosciences, Meiring-Naude Road, Pretoria, Gauteng, South Africa
5
The Matrix Biology Group, Kennedy Institute of Rheumatology, University of Oxford, 65 Aspenlea
Road, Hammersmith, London, W6 8LH, UK.
Diacylglycerol kinase (DgkA) catalyses the ATP-dependent phosphorylation of
diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to
membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gramnegative bacteria. For half a century, this 121 residue kinase has served as a model for
investigating membrane protein enzymology, folding, assembly and stability. Here we
present a case study where the use of non-standard lipids for the in meso crystallization
methodology [1-3] successfully yielded the tridimensional structure of DgkA [4].
Considerations are made on data collections strategies and difficulties during structure
determination. The crystal structures rationalize extensive biochemical and biophysical data
on the enzyme. They are, however, at variance with a published solution NMR model in that
domain swapping, a key feature of the solution form, is not observed in the crystal structures.
References
[1] Höfer N., Aragão D., Lyons J.A. and Caffrey M. (2011), “Membrane protein crystallization in lipidic mesophases.
Hosting lipid affects on the crystallization and structure of a transmembrane peptide”, Cryst Growth Des., 11:11821192.
[2] Li D., Lee J. and Caffrey M. (2011), “Crystallizing membrane proteins in lipidic mesophases. a host lipid screen”, Cryst
Growth Des,. 11:530-537.
[3] Li D., Shah S.T. and Caffrey M. (2013), “Host lipid and temperature as important screening variables for crystallizing
integral membrane proteins in lipidic mesophases. Trials with diacylglycerol kinase”, Cryst Growth Des., 13:28462857.
[4] Li D., Lyons J.A., Pye V.E., Vogeley L., Aragão D., Kenyon C.P., Shah S.T., Doherty C., Aherne M. and Caffrey M.
(2013), “Crystal structure of the integral membrane diacylglycerol kinase”, Nature, 497:521-524.
51
A tale of three structures
Megan L. O’Mara1,2, Karmen Condic-Jurkic1, Nandhitha Subramanian1, Roisin M.
McMahon3 and Alan E. Mark1,3.
1
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld, 4072,
Australia.
2
School of Mathematics and Physics, The University of Queensland, Brisbane, Qld, 4072, Australia
3
Institute of Molecular Bioscience, The University of Queensland, Brisbane, Qld, 4072, Australia
In 2009, the structure of P-glycoprotein (PDB ID 3G5U), a multidrug transporter and integral
membrane protein, was solved at 3.8 Å resolution1; however questions arose as to whether or
not this structure does indeed represent a physiological conformation. Recently, two further
structures of P-glycoprotein were solved at 3.8 Å resolution2, 3. These three structures each
have a different assignment in the amino acid register of four of the twelve helices, giving
three conflicting medium resolution structures. Using molecular dynamics simulation
techniques, we have investigated the conformation and stability of the three structures in
order to identify which structure best represents that of P-glycoprotein under physiological
conditions.
We show that in the presence of the detergent cholate, the 3G5U structure of P-glycoprotein
solved at pH 7.5 is stable. However, when incorporated into a cholesterol-enriched POPC
membrane in the presence of 150 mM NaCl the structure rapidly deforms. Only when the
simulation conditions closely match the experimental conditions under which P-glycoprotein
is transport active, is a stable conformation obtained; specifically, the presence of Mg2+,
which binds to distinct sites in the nucleotide binding domains (NBDs), and the double
protonation of the catalytic histidines (His 583 and His 1228) and His149 are required. While
the structure obtained in a membrane environment under these conditions is very similar to
that of the 3G5U crystal structure there are several key differences. The NBDs are in direct
contact, reminiscent of the open state of the Escherichia coli importer MalK. The angle
between the transmembrane domains is also increased, resulting in an outwards motion of the
intracellular loops. Notably, the structures obtained from the simulations provide a better
match to a range of experimental crosslinking data, than the original 3G5U- crystal structure
4
. This work highlights the effect that small changes in environmental conditions can have on
the conformation of a membrane protein and the importance of representing relevant
experimental conditions appropriately in modelling studies. We also examine the 2013 and
2014 P-glycoprotein structures using the simulation conditions established for the 3G5U Pglycoprotein structure and discuss the structural stability of each.
References
[1] Aller, S. G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R., Harrell, P. M., Trinh, Y. T., Zhang, Q., Urbatsch, I.
L., and Chang, G. (2009) Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding, Science
323, 1718-1722.
[2] Li, J., Jaimes, K. F., and Aller, S. G. (2014) Refined structures of mouse P-glycoprotein, Protein Science 23, 34–46.
[3] Ward, A. B., Szewczyk, P., Grimard, V., Lee, C.-W., Martinez, L., Doshi, R., Caya, A., Villaluz, M., Pardon, E.,
Cregger, C., Swartz, D. J., Falson, P. G., Urbatsch, I. L., Govaerts, C., Steyaert, J., and Chang, G. (2013) Structures of Pglycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain, Proceedings of the
National Academy of Science USA 110, 13386–13391.
[4] O'Mara, M. L., and Mark, A. E. (2012) Effect of environment on membrane protein structure: P-glycoprotein under
physiological conditions, Journal of Chemical Theory and Computation 8, 3964−3976.
52
Optimisation of G protein coupled receptor expression and
purification for structural studies
Julia K. Archbold1, Patricia M. Walden1, Fabienne A. Ferreira1, Matthew J. Sweet2, David E.
Drew3, and Jennifer L. Martin1
1
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of
Queensland, Brisbane, Queensland 4072, Australia
2
Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of
3
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-106 91 Sweden
G protein coupled receptors (GPCRs) are large transmembrane-spanning proteins that play a
pivotal role in many human diseases. There are over 800 GPCRs in our body; their role is to
sense and regulate physiological processes including sight, allergic reactions, heart beat and
brain chemistry. Because they are so critical to our health, it is no surprise that GPCRs with
abnormal function can lead to disease. Despite the interest in these proteins for drug
development, they are challenging to study experimentally as they are inherently flexible
proteins, switching between active and inactive states. When removed from their natural lipid
membrane environment, GPCRs can become unstable. These features of GPCRs have
hampered attempts to solve their crystal structures. We have screened a panel of orthologues
of a lipid binding GPCR, the LPA6 receptor, using a fluorescent GFP tagging approach [1].
We have identified the most stable, highest expressing construct of the LPA6 receptor for
expression in yeast Saccharomyces cerevisiae for future structural studies. We have also
implemented a ‘toolchest’ of fusion partners and inserted them within an intracellular loop of
the LPA6 receptor in an attempt to increase their stability and ‘crystallisability’. These fusion
partners have previously been shown to improve expression, stability and crystal packing [2].
Identification of optimal constructs and fusion partners will accelerate the structural
determination of GPCRs for structure-based drug design.
References
[1] Drew, D., et al. (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic
membrane proteins in Saccharomyces cerevisiae. Nature protocols 3, 784-798
[2] Chun, E., et al. (2012) Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors.
Structure 20, 967-976
53
SESSION 15: SCANZ 1987 PLENARY LECTURE
Chair: Bostjan Kobe, University of Queensland
Membrane protein serial femtosecond crystallography using
Petra Fromme
University of Arizona, USA
54
SESSION 16: RISING STAR PLENARY SESSION
Chair: David Turner, Monash University
The protein encapsulation and delivery mechanism of ABC toxins
Jason N. Busby1, Santosh Panjikar2,3, Michael J. Landsberg4, Mark R. H. Hurst5 and J. Shaun
Lott1
1
AgResearch Structural Biology laboratory, School of Biological Sciences, University of Auckland,
Private Bag 92019, Auckland Mail Centre, Auckland, New Zealand; 2Australian Synchrotron,
Victoria, Australia.; 3Department of Biochemistry and Molecular Biology, Monash University,
Victoria, Australia; 4Institute for Molecular Bioscience, The University of Queensland, Queensland,
Australia; 5Innovative Farming Systems, AgResearch, Lincoln Research Centre, New Zealand.
ABC toxin complexes are large, multi-subunit protein complexes produced by bacteria. They
are widespread in pathogens of insects, and play a key role in their virulence1. These
complexes typically contain three major protein components2, A, B, and C. The A
component forms a pentameric assembly that is responsible for binding to the target cell and
stimulating endocytosis. The C protein contains two distinct regions, a conserved N-terminal
region (CNTR) and a variable C-terminal toxin region (CCTR). When co-expressed with B, the
C protein is cleaved at the junction between these two regions, and all three polypeptides
remain tightly associated in a single complex.
We have studied the ABC toxin complex from a native New Zealand soil bacterium, Yersinia
entomophaga3 by X-ray crystallography and electron microscopy. We have recently
determined the crystal structure of the 243 kDa complex formed by the B and CNTR proteins.
Diffraction data were collected to ~2.5 Å at the Australian Synchrotron, and the structure was
solved using a combination of Se-SAD and a tantalum bromide cluster compound for
phasing. This structure revealed an unprecedented, large, hollow shell with a previously
unknown protein fold4. This shell is formed from a long strip of β-sheet that spirals around a
central cavity that is ~59,000 Å3 in volume. This hollow shell is believed to encapsulate the
cytotoxic CCTR and deliver it into the cytoplasm.
The C proteins contain a conserved “RHS-repeat-associated core domain”. We show that this
domain is an aspartic protease that cleaves the C protein into its two component regions, with
CCTR encapsulated inside the shell. C proteins also contain RHS (rearrangement hot-spot)
repeats, which can be found throughout bacterial species5, and also in eukaryotes6. This is
the first structure of a protein containing RHS repeats. Each individual RHS repeat
corresponds to a strand-turn-strand motif, and we predict that multiple RHS repeats will form
a long strip of β-sheet that spirals around to form a hollow shell, as seen in our structure.
RHS repeat proteins are therefore likely to be involved in the encapsulation and delivery of
proteins in many biological systems.
References
[1] Waterfield N.R., Bowen D.J., Fetherston J.D., Perry R.D. and ffrench-Constant R.H. (2001) “The tc genes of
Photorhabdus: a growing family”, Trends Microbiol., 9(4):185-191.
[2] ffrench-Constant R. and Waterfield N. (2006) “An ABC guide to the bacterial toxin complexes” Adv. Appl. Microbiol.,
58:169-183.
[3] Hurst M.R.H., Jones S.A., Binglin T., Harper L.A., Jackson T.A. and Glare T.R. (2011) “The main virulence
determinant of Yersinia entomophaga MH96 is a broad-host-range toxin complex active against insects”, J. Bacteriol.,
193(8):1966-1980.
[4] Busby J.N., Panjikar S., Landsberg M.J., Hurst M.R.H. and Lott J.S. (2013) “The BC component of ABC toxins is an
RHS-repeat-containing protein encapsulation device”, Nature, 501:547-550.
[5] Hill C.W., Sandt C.H. and Vlazny D.A. (1994) “Rhs elements of Escherichia coli: a family of genetic composites each
encoding a large mosaic protein”, Mol. Microbiol., 12(6):865-871.
[6] Minet A.D. and Chiquet-Ehrismann R. (2000), “Phylogenetic analysis of teneurin genes and comparison to the
rearrangement hot spot elements of E. coli”, Gene, 257(1):87-97.
55
The low-temperature magnetic anomaly of Ca2Fe2O5 studied by
single-crystal neutron diffraction
Josie E. Auckett1, Garry McIntyre2, Maxim Avdeev2, Hank De Bruyn1 and Chris D. Ling1
1
School of Chemistry, The University of Sydney, Sydney 2006, Australia.
The Bragg Institute, Australian Nuclear Science and Technology Organisation, Lucas Heights 2234,
Australia.
2
Ca2Fe2O5, which belongs to the Brownmillerite family of promising solid-oxide fuel cell
membrane materials, is an antiferromagnet (AFM) below TN = 720 K. A small ferromagnetic
(FM) canting perpendicular to the AFM easy axis has previously been established by physical
properties measurements, but never observed crystallographically. More intriguingly, it has
been known for some time to display an anomalous elevation in magnetic susceptibility for
60 K < T < 140 K. [1] Based on measurements performed with small oriented single crystals,
Zhou et al. [2] proposed that this anomaly was due to a reorientation of the spins from the
crystallographic a axis to the c axis below 40 K, with a region of minimal magnetocrystalline
anisotropy in the anomalous temperature interval.
In order to test this, we grew a very large (~1 cm3) single crystal by the floating-zone method
and collected neutron Laue diffraction data, against which we refined both the atomic and
magnetic structures of Ca2Fe2O5 between 10 K and 300 K. We designed and built an ad hoc
sample mount to apply a small (~35 Oe) magnetic field to the sample, ensuring perfect
consistency with the magnetic susceptibility data, which were collected in a comparably
small field. Our refinements against both zero-field and in-field diffraction data reproduce the
G-type AFM structure of Ca2Fe2O5 excellently at room temperature, including the FM
canting which we have refined to statistical significance for the first time. We can also show
that in the intermediate temperature interval (T = 100 K), the spins are slightly less wellordered due to competing sublattice interactions. However, careful examination of the data
reveals that the material is still best described by the room-temperature magnetic structure at
all measured temperatures – i.e., the spin-reorientation hypothesis is incorrect.
References
[1] Maljuk A., Strempfer J. and Lin C.T. (2003) “Floating zone growth and characterization of Ca2Fe2O5 single crystals”, J.
Cryst. Growth, 258:435-440.
[2] Zhou H.D. and Goodenough J.B. (2005) “Rotation of magnetocrystalline easy axis in Ca2Fe2O5”, Solid State Sci.,
7:656-659.
56
The structure and dynamics of rotary ATPases
Alastair G. Stewart1,2, Lawrence K. Lee1,2, Mhairi Donohoe1,2, Jessica J. Chaston1,2 and
Daniela Stock1,2
1
Structural Biology, The Victor Chang Cardiac Research Institute, NSW 2010, Australia.
Faculty of Medicine, The University of New South Wales, Australia
2
Rotary ATPases are central components of the
biological energy conversion machinery found across
all known forms of life [1]. They are large enzyme
complexes that couple ATP hydrolysis/synthesis with
ion translocation via a rotary catalytic mechanism.
Their peripheral stalks (see figure insert) are essential
components that counteract torque generated by the
enzyme during ATP synthesis or hydrolysis. Using
X-ray crystallography we are able to present two
atomic models of the peripheral stalk of the A-type
ATPase/synthase from the bacterium Thermus
thermophilus [2,3]. These structures were obtained
from two different crystal forms and have
significantly different conformations that give important insight into their function. The
structures contain a heterodimeric right-handed coiled coil, a protein fold that has not been
observed previously. Fitting of the crystal structures into a low resolution electron
microscopy reconstruction of the intact complex enabled the precise location of the peripheral
stalk to be established. By comparing the structures in combination with normal mode and
Fourier analysis, it was possible to show that the unusual right-handed coiled coil has evolved
to provide a structure that is strongest in the direction of rotation, complementing its
mechanical function.
References
[1] Stewart A.G., Sobti M., Laming, E. and Stock, D. (2014) “Rotary ATPases - dynamic molecular machines”, Current
Opinion in Structural Biology, 25:40.
[2] Lee, L.K.., Stewart A.G., Donohoe M., Bernal R.A. and Stock D.(2010) “The structure of the peripheral stalk of T.
thermophilus H+-ATPase/synthase”, Nature Structural & Molecular Biology, 17:373.
[3] Stewart A.G., Lee L.K.., Donohoe M., Chaston J.J. and Stock D.(2012) “The dynamic stator stalk of rotary ATPases”,
Nature Communications, 3:687.
57
New structural insights into receptor activation and assembly in the
beta common cytokine family
Sophie E. Broughton,1 Timothy R. Hercus,2 Tracy L. Nero,1 Urmi Dhagat,1 Barbara J.
McClure2, Mara Dottore2, Angel F. Lopez2 and Michael W. Parker,1,3
1
Australian Cancer Research Foundation Rational Drug Discovery Centre, St. Vincent’s Institute of
Medical Research, 9 Princes St, Fitzroy, Victoria
2
Division of Human Immunology, the Centre for Cancer Biology, SA Pathology, Frome Road,
Adelaide, South Australia
3
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology
Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria, Australia
Interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5
are important cytokines that control the production and function of myeloid cells and
dendritic cells. Over-expression of these cytokines or their receptors can lead to chronic
inflammatory diseases such as rheumatoid arthritis and asthma, and cancers such as myeloid
leukemias.
All three cytokines signal through specific heterodimeric receptors consisting of major
binding and cytokine-specific α-receptors, and a signalling subunit βc, shared by all three
cytokines. Recent crystal structures of the GM-CSF receptor ternary complex (GMCSF:GMRα:βc) and the IL-5 binary complex (IL-5:IL5Rα), together with associated
structure-function studies have significantly enhanced our understanding of how these
receptors recognise cytokines and signal across cell membranes [1, 2]. We now report
previously unresolved 3D structures of the βc family members based on X-ray diffraction,
including for the first time all three extracellular domains of the GMRα receptor. Overall the
GMRα structure shows a striking resemblance to the related IL5Rα and IL13Rα receptors;
however, the position of the N-terminal domains of the α-receptors appear to differ,
suggesting that the N-terminal domain may play a cytokine-specific role in cell signalling. In
addition, mutagenesis, ligand binding and functional studies have been used to examine
residues in the cytokine and α-receptor subunit of the βc cytokine family members that define
the binding interfaces of these complexes, and further elucidate key residues involved in
higher order complex assembly. These studies enable us to identify differences or similarities
that define the specificity and affinity of these ligand:receptor interactions that contribute to
downstream signalling by these cytokines. Importantly, these structures provide the
opportunity for structure-based approaches for the discovery of novel and disease specific
therapeutics.
References
[1]
[2]
Hercus, T. R., Thomas, D., Guthridge, M. A., Ekert, P. G., King-Scott, J., Parker, M. W., and Lopez, A. F. (2009) The
granulocyte-macrophage colony-stimulating factor receptor: linking its structure to cell signalling and its role in
disease. Blood 114:1289-1298
Broughton S. E., Dhagat U., Hercus T. R, Nero T. L., Grimbaldeston M. A., Bonder C. S., Lopez A. F., and Parker M.
W (2009) The GM-CSF/IL-3/IL-5 cytokine receptor family: from ligand recognition to initiation of signalling.
Immunol Rev 250:277-302.
58
Structural basis of disease resistance in flax against flax rust
Thomas Ve1, Simon Williams1, Maud Bernoux2, Ann-Maree Catanzariti3, Motiur Rahman4
David Jones3, Jeffrey G. Ellis2, Peter Anderson4 Peter N. Dodds2, Bostjan Kobe1
1
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072,
Australia, 2CSIRO Plant Industry, Canberra, Australia, 3Plant Science Division, Research School of
Biology, Australian National University, Canberra, Australia, 4School of Biological Sciences,
Flinders University, Adelaide, SA 5001, Australia
Plant diseases are a major issue for economical important crops worldwide. Plant immunity is
triggered by the recognition of a pathogen effector protein by a plant resistance (R) protein,
leading to the activation of plant defences, which often culminate in a localized cell death
response [1]. The R proteins can be divided into a few conserved families, while the effectors
are diverse in both sequence and structure, and have roles in virulence. Recognition of
effectors by R proteins, and the subsequent activation and downstream signaling events, are
poorly understood at the molecular and structural level. We have used the interaction between
flax and the fungal pathogen, flax rust, as a model system to characterize this process. The
flax- R proteins consist of a central nucleotide-binding (NB) domain, a C-terminal leucinerich-repeat (LRR) domain, and an N-terminal Toll-interleukin-receptor like (TIR) domain.
The LRR domains of flax R proteins are involved in direct interaction with corresponding
flax-rust effectors, while the NB and TIR domains have roles in activation and signalling,
respectively.
Here, we report the first crystal structure of a TIR domain from a plant R protein (L6) at 2.3
Å resolution [2]. The structure reveals important differences from the structures of
mammalian and bacterial TIR domains. Analysis of the structure combined with site-directed
mutagenesis suggests that TIR domain self- association is a requirement for immune
signaling, and reveals distinct surface regions involved in self-association, signaling, and
autoregulation. We have also determined the crystal structure of the secreted flax rusteffector protein AvrM. AvrM can internalize into plant cells and in the absence of the
pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance
protein M in flax [3]. The structure reveals an L-shaped fold consisting of a tandem
duplicated four-helix motif, which displays similarity to the WY domain core in oomycete
effectors [4]. Our functional analysis of AvrM reveals that a conserved hydrophobic surface
patch is required for internalization into plant cells, whereas the C-terminal coiled-coil
domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface
charges, and mutations that abrogate PIP binding have no significant effect on internalization,
suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant
membrane.
Our results bring us a step closer to understanding the molecular basis for the disease
resistance process in plants, which is a prerequisite for the future engineering of novel
resistance specificities into commercially important crops.
References
[1] Dodds P.N., and Rathjen J.P. (2010), “Plant immunity: towards an integrated view of plant–pathogen interactions”,
Nature Reviews Genetics 11: 539-548.
[2] Bernoux M, and Ve T. et al. (2011), “Structural and functional analysis of a plant resistance protein TIR domain reveals
interfaces for self-association, signaling, and autoregulation”, Cell Host Microbe 9: 200-211.
[3] Catanzariti A.M. et al. (2010), “The AvrM effector from flax rust has a structured C-terminal domain and interacts
directly with the M resistance protein”. Mol Plant Microbe Interact 23: 49-57.
[4] Ve T. et al. (2013), ‘Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry
and effector-triggered immunity’, PNAS 110 (43): 17594-17599.
59
Insights into vibrational stark effect from X-ray charge density
analysis of supramolecular host-guest complexes
Sajesh P. Thomas, Rebecca O. Fuller, Alexandre N. Sobolev, Philip A. Schauer, Simon
Grabowsky, George A. Koutsantonis and Mark A. Spackman
School of Chemistry and Biochemistry, The University of Western Australia, Crawley,WA 6009.
The Vibrational Stark Effect (VSE), the effect of an electric field on the vibrational spectrum,
has been utilized extensively to probe the local electric field in the active sites of enzymes
[1]. For this reason the electric field induced by a supramolecular host system upon its guest
molecules is a topic of special interest due to its implications for various biological processes.
Although the host-guest chemistry of clathrates and crown ether complexes is of fundamental
importance in supramolecular chemistry, many of these simple systems have yet to be
explored in detail using modern techniques [2]. In this context, the results from the
experimental charge density analyses of the inclusion complexes of chloro- and
fluoroacetonitrile and formamide with 18-crown-6 host molecules will be presented. The
charge density models provide estimates of the molecular dipole moment enhancement and
the electrostatic contribution associated with the host-guest interactions. Accurate mapping of
the electron density using the multipolar model also provides an estimate of the electric field
experienced by the guest molecules, and using this field the VSE in the nitrile (-C≡N) and
carbonyl (C=O) stretching frequencies of the guest molecules are estimated via quantum
chemical calculations on the guest molecules. The results of these calculations indicate
notable bond elongation in C≡N and C=O bonds due to the field. The electronic polarization
along these covalent bonds induced by the electric field manifests as significant red shifts in
vibrational frequencies. These observations are substantiated by FT-IR experiments and thus
quantitatively establish the phenomenon that could be termed as the “supramolecular Stark
effect” in crystal environment.
References
[1] (a) Park E.S. and Boxer S.G. (2002) “Origins of the sensitivity of molecular vibrations to electric fields: carbonyl and
nitrosyl stretches in model compounds and proteins”, J. Phys. Chem. B, 106:5800-5806. (b) Suydam I.T., Snow C.D.,
Pande V.S. and Boxer S.G. (2006) “Electric fields at the active site of an enzyme: direct comparison of experiment with
theory”, Science, 313: 200.
[2] Clausen H.F, Chen Y.S., Jayatilaka D., Overgaard, J., Koutsantonis G.A., Spackman M.A. and Iversen B.B. (2011)
“Intermolecular interactions and electrostatic properties of the β-hydroquinone apohost: implications for supramolecular
chemistry”, J. Phys. Chem. A, 115: 12962–12972.
60
Chair: Jennifer L Martin, University of Queensland
Crystallography and climate change: The temperature-dependence
of biological rates
Vic Arcus
Waikato University, New Zealand
61
POSTER SESSION
Vasorelaxant activity of Canavalia grandiflora seed lectin: A
structural analysis
Maria Júlia Barbosa Bezerra1, Ito Liberato Barroso-Neto IL1, Rafael da Conceição Simões1,
1
1
Bruno Anderson Matias Rocha , Francisco Nascimento Pereira-Junior , Vinicius José Silva
Osterne1, Kyria Santiago Nascimento1, Celso Shiniti Nagano2, Plinio Delatorre3, Maria Gonçalves
Pereira4, Alana Freitas Pires4, Alexandre Holanda Sampaio2, Ana Maria Assreuy4, Benildo
Sousa Cavada1
1
Laboratory of Biologically Active Molecules, Federal University of Ceara, Fortaleza, Brazil
Department of Fisheries Engineering, Federal University of Ceara, Brazil
3
Department of Molecular Biology, Federal University of Paraiba, Brazil
4
Institute of Biomedical Sciences, State University of Ceara, Brazil
2
Lectins are comprised of a large family of proteins capable of the specific and reversible
recognition of carbohydrates1. Legume lectins, the most studied plant lectins, show high
structural similarity, but with modifications that imply a variation in the intensity of some
biological activities2-7. In this work, the primary and tertiary structures of Canavalia
grandiflora (ConGF) were determined. ConGF, a lectin isolated from C. grandiflora seeds, is
able to induce relaxant activity in rat aortic rings. The complete sequence of ConGF
comprises 237 amino acids. This particular protein has primary sequence variations
commonly found in lectins from Dioclea and Canavalia genera. The protein structure was
solved at 2.3Å resolution by X-ray crystallography. An X-Man molecule was modeled into
the carbohydrate recognition domain. Still, ConGF (30 and 100 µgmL(-1)) elicited 25% of
vasorelaxation (IC50=34.48±5.07µgmL(-1)) in endothelialized aortic rings. A nonselective
inhibitor of nitric oxide blocked ConGF relaxant effect, showing mediation by nitric oxide.
Key distances between ConGF carbohydrate recognition domain residues were determined in
order to explain this effect, in turn revealing some structural aspects that could differentiate
lectins from the Canavalia genera with respect to different efficacy in vasorelaxant effect.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
Cavada B.S., Barbosa T., Arruda S., Grangeiro T.B. and Barral-Netto M. (2001), Curr. Protein Pept. Sci. 2:123–
135.
Sanz-Aparício J., Hermoso J., Granjeiro T.B., Calvete J.J.and Cavada B.S. (1997) FEBS Lett. 4: 114–118.
Bezerra E.H.S., Rocha B.A.M., Nagano C.S., Bezerra G.D., Moura T.R., Bezerra M.J.B., Benevides R.G., Sampaio
A.H., Assreuy A.M.S., Delatorre P. and Cavada B.S. (2011) Biochem. Biophys. Res. Commun. 408: 566–570.
Cavalcante T.T.A., Rocha B.A.M., Carneiro V.A., Arruda F.V.S., Nascimento A.S.F., Sá N.C., Nascimento K.S.,
Cavada B.S. and Teixeira E.H. (2011), Molecules 16: 3530– 3543.
Rocha B.A.M., Delatorre P., Oliveira T.M., Benevides R.G., Pires A.F., Sousa A.A., Souza L.A., Assreuy A.M.S.,
Debray H., Azevedo-Junior W.F., Sampaio A.H. and Cavada B.S. (2011), Biochimie 9: 806–816.
Nóbrega R.B., Rocha B.A.M., Gadelha C.A.A., Santi-Gadelha T., Pires A.F., Assreuy A.M.S., Nascimento K.S.,
Nagano C.S., Sampaio A.H., Cavada B.S. and Delatorre P. (2012), Biochimie 94: 900–906.
Bezerra M.J.B., Rodrigues N.V.F.C., Pires A.F., Bezerra G.A., Nobre C.B., Alencar K.L.L., Soares P.M.G.,
Nascimento K.S., Nagano C.S., Martins J.L., Gruber K., Sampaio A.H., Delatorre P., Rocha B.A.M., Assreuy A.M.S.
and Cavada B.S. (2013), Int. J. Biochem. Cell Biol. 45: 807–815.
62
Characterisation of adenylosuccinate synthetase from the fungal
pathogen Cryptococcus neoformans
Ross Blundell1, Simon J. Williams1,2, Carl A. Morrow1, Bostjan Kobe1,2 and James A. Fraser1
1
Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences,
The University of Queensland, Brisbane, QLD, Australia
2
Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australis
With increasingly large immunocompromised populations around the world, opportunistic
fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and
mortality; C. neoformans is estimated to be responsible for over 600000 deaths every year.
Fungal infections are difficult to treat owing to the similarities of their eukaryotic physiology
to that of humans. The limited antifungal agents available tend to target the few differences
between the two systems. To combat the current scarcity of antifungal therapeutic agents,
research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is
a crucial enzyme in the ATP biosynthetic pathway, catalyzing the formation of
adenylosuccinate from inosine monophosphate and aspartate. We investigated the potential of
this enzyme as an antifungal target, finding that loss of function results in adenine auxotrophy
in C. neoformans, as well as changes in the production of major virulence factors and
virulence in an animal model. Cryptococcal AdSS was also expressed and purified from E.
coli and the enzyme’s crystal structure solved. Combined with enzyme kinetic studies, this
structural information enables comparisons with human homologues to facilitate future in
silico screens for novel antifungal compounds.
63
Crystallography365 and Crystals in the City: IYCr 2014 activities.
H.E. Maynard-Casely1 and N. Sharma2
1
Bragg Institute, Australian Nuclear Science and Technology Organisation, Kirrawee DC, NSW,
2234, Australia
2
Department of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia
Reflecting the strong heritage of crystallographic research in Australia, we wish to present
two of the public outreach projects that are underway to celebrate International Year of
Crystallography 2014.
A project that is already up and running is Crystallography365 - Blogging a crystal structure
a day at http://crystallography365.wordpress.com/. Gathering a group of, principally students
and early career researchers based in Australia, each day during 2014 a different crystal
structure will be presented and described. The goals of the project is to present the wide
range of uses crystal structures have to a broad spectrum of sciences, and to provide an outlet
for this group of scientists to engage with International Year of Crystallography.
The other (hopefully bigger) project, Crystals in the city, will run 9th-30 August 2014
(coinciding with National Science Week in Australia). A partnership between ANSTO and
University of New South Wales, it will bring a public display of 6 person-size crystal
structure models exhibited in cities around Australia. The goal is that the crystal structures
will ‘reflect’ their surroundings and instil pride among the public in the crystallographic
achievements of Australian science. Accompanying the exhibition will be website, where the
public can find more about each of the structures and students can learn of studying
opportunities. The project will also unite a host of supporters and sponsors; universities,
museums and crystallographic groups.
64
Solvent exchange in a hierarchically assembled metal-organic
framework
Aidan J. Brock and Jack K. Clegg
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia 4072,
Australia.
The concept of hierarchical self-assembly has been proposed as a new method for the
development of metal-organic frameworks (MOFs) with large pore volume and well-defined
host-guest chemistry. In this process, a discrete metallo-supramolecular complex is prepared.
This complex contains free sites for further coordination, in the form of unsaturated metal
centres or additional ligands, which can then be used to extend the complex into a polymeric
structure. Few three-dimensional examples of hierarchically-assembled MOFs have been
reported. An early example of a 3D hierarchically-assembled MOF was reported by Clegg
and coworkers in 20101, in which a trinuclear metallocycle based on copper(II) ions and the
bis-β-diketonato ligand 1,1'-([1,1'-biphenyl]-4,4'-diyl)bis(4,4-dimethylpentane-1,3-dione) was
extended into a 3D MOF by the addition of hexamethylenetetramine (hmt), which acts as a 3connecting ligand be coordinating to three unsaturated copper(II) centres. This MOF was
shown to have a large pore volume, containing 90 disordered THF molecules in the unit cell.
Figure 1 – Hierarchically assembled Cu(II) MOF (solvent not shown). The trinuclear subunits can be clearly
observed.
We have now shown that it is possible to exchange these THF molecules with a variety of
other solvents by soaking the single crystals in the desired solvent. The unit cell of the MOF
has been observed to vary significantly according to the volume of the solvent molecule. In
addition to this, ordered solvent molecules have been crystallographically observed in a
number of crystals, including those containing 1,4-dioxane, N,N-dimethylformamide,
chloroform and dichloromethane. The MOF has also been observed to have a very large
thermal expansion coefficient.
References
[1] Clegg J. K., Iremonger S. S., Hayter M. J., Southon P. D., Macquart R.B., Duriska M. B., Jensen
P., Turner P., Jolliffe K.A., Kepert, C. J., Meehan, G.V. and Lindoy, L. F. (2010), Angew Chem
Int Ed, 49.
65
Density functional calculations of electron energy loss data and
inelastic mean free paths in elemental and binary materials
School of Physics, University of Melbourne,Parkville VIC 3010, Australia
Density functional theory is used to determine joint density of states and optical energy loss
data for elemental metals and compounds. A novel transform algorithm is then employed to
produce electron energy loss spectra and inelastic mean free paths. These results are
compared with recent high profile experimental measurements in the low-energy region of
strong plasmon resonance.
We present calculations of electron energy loss spectra and inelastic mean free paths
determined using transformed optical loss data from density functional theory. Our novel
approach enables investigation of plasmon excitation and electron scattering without the use
of empirical fitting parameters or experimental data - common requirements of previous
theory [1]. We consider elemental metals copper, molybdenum, zinc and selenium, and the
binary semiconductor zinc selenide.
Results are compared with recent high precision measurements of the inelastic mean free path
[2, 3] derived from x-ray absorption experiments [4, 5]. Broadening mechanisms in the
momentum-dependent energy loss function, and the energy evolution of plasmon peaks are
investigated as potential sources of error in established theory. We consider the effects of
crystal structure on the energy loss spectrum, and critically investigate the correlation
between inelastic scattering in polyatomic systems and their corresponding elemental
systems. This work focuses on energies below 100 eV – the region of strong plasmon
resonance – where published loss data is typically highly uncertain and, in many cases,
incomplete.
References
[1]
[2]
[3]
[4]
[5]
Tanuma, S., Powell C.J. and Penn D.R. (2011), Surf. Interface Anal. 43:689
Bourke, J.D. and Chantler C.T. (2010), Phys. Rev. Lett. 104: 206601
Chantler C.T. and Bourke J.D. (2010), J. Phys. Chem. Lett. 1: 2422
Glover J.L., Chantler C.T., Barnea Z., Rae N., Tran C.Q., Creagh D.C., Paterson D. and Dhal B.B. (2008), Phys. Rev.
A 78: 052902
de Jonge M.D., Tran C.Q., Chantler C.T., Barnea Z., Dhal B.B., Cookson D.J., Lee W. and Mashayekhi A. (2005),
Phys. Rev. A, 71: 032702
66
Structural insights into Bak activation and oligomerisation
Jason M. Brouwer1,2, Adeline Y. Robin1,2, Geoff V. Thompson1,2 , Ahmad Z. Wardak1,2, Peter
M. Colman1,2 and Peter E. Czabotar1,2
1
Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052,
Australia
2
Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3052, Australia
Apoptotic stimuli activate and oligomerise the pro-apoptotic proteins Bak and Bax resulting
in mitochondrial outer membrane permeabilisation and subsequent cell death. This activation
can occur when certain BH3-only proteins directly interact with Bak and Bax. A recent
crystal structure by Czabotar et al. (2013) revealed a novel conformational change for Bax
upon activation by BH3-only peptides. Distinguishing characteristics of BH3-only proteins
capable of directly activating Bax were also elucidated. Here we describe complementary
studies on the related protein Bak. We identify specific BH3-only peptides capable of
inducing Bak dimerisation and describe crystal structures that provide key insights into Bak
activation and oligomerisation. These structures demonstrate that Bak undergoes similar
conformational changes upon activation to those observed with Bax. Altogether our results
confirm an analogous mechanism for activation and dimerization of Bak and Bax in response
to BH3-only peptides.
References
[1] Czabotar P.E., Westphal D., Dewson G., Ma, S., Hockings C., Fairlie W.D., Le E.F., Yao, S.,
Robin A.Y., Smith B.J., Huang D.C.S., Kluck R.M., Adams J.M. and Colman, P.M. (2013),
“Structural transitions activating bax for apoptosis”, Cell, 152 (3): 519-31
67
Structural and dynamic studies of PPARγ agonism
Laura Marrewijk1, David Marciano2, Ted Kamenecka2, Patrick Griffin2 and John B. Bruning1
1
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia, 5005
Department of Molecular Therapeutics, 130 Scripps Way, The Scripps Research Institute, Jupiter,
Florida, USA, 33458
2
Despite the clinical benefit of diabetic therapeutics such as the TZD (thiazolidinedione)
compound class, their use has been associated with adverse effects including weight gain,
increased adipogenesis, renal fluid retention, plasma volume expansion and possible
increased incidence of cardiovascular events. The nuclear receptor PPARγ (Peroxisome
Proliferator-Activated Receptor) is the target of antidiabetic compounds such as the full
agonist TZDs. Partial agonists, however, have been shown to have lessened side effects and
act through different structural mechanisms than full agonists. We have undertaken a large
scale structural study to identify the mechanism of partial agonism of PPARg. By means of
X-ray crystallography and Hydrogen Deuterium Exchange (HDX) experimentation on a large
test set of partial agonists and full agonists we are dissecting the epitope specific signatures of
these classes of compounds. Initial results suggest that helix 12, helix 11, and the beta sheet
region are specifically modulated in a differential manner between full and partial agonists of
PPARg. These compounds show potent antidiabetic activity while not causing the fluid
retention and weight gain that are serious side effects of many of the PPARγ drugs.
68
In search of infinite solutions; the inverse problem in small-angle Xray scattering
Lachlan W. Casey1,2, Alan E. Mark1,2 and Bostjan Kobe1,2,3
1
School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia 4072, Australia.
Institute for Molecular Bioscience, University of Queensland, St Lucia 4072, Australia.
3
Australian Infectious Diseases Research Centre, University of Queensland, St Lucia 4072, Australia.
2
Small-angle X-ray scattering (SAXS) has emerged as a powerful and versatile complement to
macromolecular crystallography, but also one requiring careful interpretation1. Here, we have
applied SAXS to the classical nuclear import receptor, importin-β, in a case study that
illustrates both the limitations and the power of the technique. The protein possesses a
solenoid architecture which permits large-scale flexibility while retaining tertiary structure,
and has been crystallized in a number of forms2,3. Its spring-like extension can be
approximated by a single coordinate4, the radius of gyration (Rg), and we exploit this to
examine both the behavior of the protein and the robustness of ensemble modeling from
SAXS, molecular dynamics and crystal structures.
We find that scattering data for apo importin-β is best described by an ensemble of
conformers dominated by structures with Rgs within 3 Å of that of the RanGTP-bound
structure. However, we extend our analysis to examine the effects of limited conformational
sampling on the selection procedure. We find that ensemble fitting algorithms are able to
adjust for large perturbations in the conformational pool, and that for this system, mutuallyexclusive conformational pools can be identified that each lead to consistent restorations of
the data.
In light of this, we highlight the risk of over-interpreting scattering data through ensemble
modeling, and suggest that identifying a dataset's tolerance for varying models is a key step
in analysis when restoring atomic structures from scattering data.
References
[1]
[2]
[3]
[4]
Trewhella J., Hendrickson W.A., Kleywegt G.J., Sali A., Sato M., Schwede T., Svergun D.I., Tainer J.A., Westbrook
J. and Berman H.M. (2013) "Report of the wwPDB Small-Angle Scattering Task Force: data requirements for
biomolecular modeling and the PDB", Structure, 21:875-81.
Liu S.M. and Stewart M. (2005) "Structural basis for the high-affinity binding of nucleoporin Nup1p to the
Saccharomyces cerevisiae importin-beta homologue, Kap95p", Journal of molecular biology, 349:515-25.
Forwood J.K., Lange A., Zachariae U., Marfori M., Preast C., Grubmüller, H., Stewart M., Corbett A. H. and Kobe,
B. (2010) "Quantitative structural analysis of importin-β flexibility: paradigm for solenoid protein structures",
Structure, 18:1171-83.
Kappel C., Zachariae U., Dölker N. and Grubmüller, H. (2010) “An unusual hydrophobic core confers extreme
flexibility to HEAT repeat proteins”, Biophysical journal, 99:1596-603
69
Structural basis of binding specificity between nuclear receprtor:
importin-α and non-classica nuclear localization signals
Chiung-Wen Chang1, 2, Rafael L.M. Couñago1,2, Simon J. Williams1, 2, Mikael Bodén1, 3 and
Boštjan Kobe1, 2
1
School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience, University of
Queensland, Brisbane, Qld 4072, Australia.2 Australian Infectious Diseases Research Centre,
University of Queensland, Brisbane, Qld 4072, Australia.3 School of Information Technology and
Electrical Engineering, University of Queensland, Brisbane, Qld 4072, Australia.
In the classical nuclear import pathway, the specific recognition between the nuclear receptor
(importin-α) and the nuclear localization signals (NLSs) plays an essential role on facilitating
the cargo import process. Importin-α has two separate NLS-binding sites (the major and the
minor sites), accommodate NLSs, comprising of one (monopartite) or two clusters (bipartite)
of basic residues connected by a 10 - 12 residue linker. The major NLS-binding site is the
preferential binding site for most of the monopartite NLSs characterized to date. By screening
random peptide libraries using importin-α variants as bait, the bound NLS sequences could be
divided into six classes1. The class-3 minor site-specific NLSs and class-5 plant-specific
NLSs feature a shorter basic cluster. The molecular basis of the specific binding between
these non-classical NLSs and importin-α was not known and in particular, there was a lack of
crystal structures of plant importin-α. Here, we present the first crystal structure of plant
importin-α, and explain the differential binding specificity between the class-5 plant-specific
NLSs and importin-α variants2. The binding conformation of the class-3 minor site-specific
NLSs features an α-helical turn, that is distinct from the other NLSs reported structurally3.
Comparative bioinformatic screens not only indicate both plant-specific and minor sitespecific NLSs are much less prevalent than the classical NLSs, but also reveal a greater
prevalence of these two classes of non-classical NLSs in rice the proteome, compared to the
others from yeast, mammals, and even other plant species4. Together, our data can help to
characterize novel proteins containing non-classical NLSs destined for the cell nucleus by the
classical nuclear import pathway.
References:
[1]
[2]
Kosugi S., Hasebe M., Matsumura N., Takashima H., Miyamoto-Sato E., Tomit M. and Yanagawa H. (2009). “Six
classes of nuclear localization signals specific to different binding grooves of importin alpha”, J Biol Chem., 2;
284(1): 478-85.
Chang, C.-W., Couñago, R.. Mi., Williams, S.J., Bodén M. and Kobe B. (2012). “Crystal Structure of Rice Importinα and Structural Basis of Its Interaction with Plant-Specific Nuclear Localization Signals”.The Plant Cell,vol. 24 no.
12 5074-5088.
[3]
Chang, C.-W., Couñago, R. Mi., Williams, S. J., Bodén M. and Kobe B. (2013). “Distinctive conformation of minor
site-specific nuclear localization signals bound to importin-α”. Traffic, 14 (11): 1144-54.
[4]
Chang, C.-W., Couñago, R. . Mi., Williams, S. J., Bodén M. and Kobe B. (2013).“The distribution of different
classes of nuclear localization signals (NLSs) in diverse organisms and the utilization of the minor NLS-binding site
inplant nuclear import factor importin-α”. Plant Signaling & Behavior, 8 (10), pii:e25976.
70
High accuracy calibration of a synchrotron x-ray beam using
powder diffraction
L.J.Tantau,1 M.T.Islam,1 C.T. Chantler,1 N.A.Rae,1 A.T.Payne,1 C.Q.Tran2 and M.H.Cheah3
1. School of Physics, University of Melbourne, Parkville, Victoria 3010, Australia; 2. La Trobe
University; 3. University of Queensland
Accurate knowledge of the energy of an x-ray beam produced by a synchrotron source is
important in many fields involving synchrotron radiation, including but not limited to: x-ray
Absorption Fine Structure (XAFS), many forms of diffraction and crystallography. We
present our method to determine the energy of an x-ray beam with measurements made with
the large powder diffractometerBigDiff at the Australian National Beamline Facility (ANBF)
[1]. The large diffractometer permits multiple powder patterns to be taken on single image
plates courtesy of it's Debye-Scherrer camera setup [2]. Full powder patterns are able to be
collected in a short amount of time, and even coarsely spaced energy measurements can
provide accuracy energy calibration over a large energy range. We show that a broad range of
experimental systematics are able to be analysed using our technique [3], providing the high
accuracy required for many fields using x-rays produced by a synchrotron source [4].
References
[1]
[2]
[3]
[4]
Barnea Z., Creagh D.C., Davis T.J., Garrett R.F., Janky S., Stevenson A.W. and Wilkins S.W. (1992) “The
Australian diffractometer at the photon factory”, Review of Scientific Instruments, 63(1):1069-1072.
Garrett R.F., Cookson D.J., Foran G.J., Sabine T.M., Kennedy B.J. and Wilkins W.2. (1995), “Powder diffraction
using imaging plates at the Australian National Beamline Facility at the Photon Factory”. Review of Scientific
Instruments, 66(2):1351-1353.
Rae N.A., Islam M.T., Chantler C.T. and de Jonge M.D. (2010), E”nergy determination of synchrotron X-ray beam
energy in the high energy region of 38-50 keV using powder diffraction patterns of the standard powder Si640b”
Nuclear Instruments and Methods in Physics Research Section A:Accelerators, Spectrometers, Detectors and
Associated Equipment, 619(1-3):147-149.
Tantau L.J., Chantler C.T., Payne A.T., Islam M.T., Tran C.Q., Best S.P., Cheah M.C. (2014), “High-accuracy X-ray
energy determination and comparison from LaB6 powder diffraction pattern for synchrotron XAFS and XRD
calibration”, Rad. Phys. Chem. 95: 73-77
71
The intersection of two central cellular trafficking pathways: the
binding of SNX27 to the retromer complex
Thomas Clairfeuille1,a, Matthew Gallon2,a, Caroline Mas1, Peter J. Cullen2 and Brett M.
Collins1
1
Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland, 4072,
Australia.
2
The Henry Wellcome Integrated Signalling Laboratories, School of Biochemistry, University of
Bristol, Bristol BS8 1TD, UK.
a. These authors contributed equally to this work.
The soluble cargo recognition core trimer Vps26-Vps29-Vps35 of the mammalian retromer
complex binds to transmembrane receptors embedded in the endosomal membrane.
Interactions with the SNX-BAR subcomplex and the Rab7 GTPase, among other proteins,
partitions retromer into tubular sub-domains where cargo sorting events take place [1].
Initially characterised in retrograde trafficking of cargo proteins from endosomes to the transGolgi network (TGN) [2], multiple studies have established the crucial role of retromer in
recycling of endocytic cargo to the plasma membrane [3]. Our recent proteomics analysis
identified a number of important cell surface proteins whose trafficking is altered upon the
loss of sorting nexin 27 (SNX27) or Vps35. More than eighty proteins appear to depend on
the SNX27-retromer complex for endosome-to-plasma membrane recycling. Among these
proteins are biologically critical cargoes that possess a C-terminal PSD95-disc largezonula occludens (PDZ)-sorting signal able to bind to the PDZ domain of SNX27, such as the
β2-adrenergic receptor (β2-AR) and glucose transporter 1 (GLUT1) [4].
In this study, we demonstrate that SNX27 conservatively binds to both Vps26 paralogues,
Vps26A and Vps26B, through the structurally unique features of the PDZ domain. We report
the first crystal structure of a PDZ domain bound to a different protein, as well as the first
structural evidence of the retromer cargo-recognition complex binding to another protein
partner. By demonstrating the ability of SNX27 to independently bind to PDZ ligands whilst
being engaged with Vps26, our biochemical and trafficking studies confirm SNX27 as a
cargo adaptor for retromer, thus linking a plethora of PDZ domain-mediated sorting events to
this recycling pathway. Perturbation of the SNX27-Vps26 association abolished recycling of
the GLUT1 receptor from endosomes to the plasma membrane, confirming that the binding
of SNX27 to the VPS-sub-complex is required for retromer-mediated endosome-to-plasma
membrane recycling. In summary, we thoroughly dissect a novel mechanism of protein
binding by a PDZ domain and provide molecular insight into the biologically critical
endocytic recycling of over a hundred proteins via the SNX27-retromer-mediated pathway.
References
[1] Cullen P.J. and Korswagen H.C. (2012) “Sorting nexins provide diversity for retromer-dependent trafficking events”,
Nat Cell Biol, 14(1): 29-37.
[2] Arighi C.N., Hartnell L.M., Aguilar, R.C., Haft C.R. and Bonifacio J.S. (2004) “Role of the mammalian retromer in
sorting of the cation-independent mannose 6-phosphate receptor”, J Cell Biol, 165(1):123-33.
[3] Temkin P., Lauffer B., Jager .S, Cimermancic P., Krogan N.J. and von Zastrow, M. (2011) “SNX27 mediates retromer
tubule entry and endosome-to-plasma membrane trafficking of signalling receptors”, Nat Cell Biol, 13(6): 715-21.
[4] Steinberg F., Gallon M., Winfield M., Thomas .E.C, Bell A.J., Heesom K.J., Tavare J.M. and Cullen P.J. (2013) “A
global analysis of SNX27-retromer assembly and cargo specificity reveals a function in glucose and metal ion
transport”, Nat Cell Biol, 15(5): 461-71.
72
Structural investigations into the control of pro-apoptotic Bcl-2
family proteins
Angus D Cowan1,2, Peter E Czabotar1,2 and Peter M Colman1,2
1
Structural Biology Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade,
Parkville, Victoria 3052, Australia.
2
Department of Medical Biology, University of Melbourne, Parkville, Victoria 3052, Australia.
Programmed cell death, or apoptosis, is responsible for the controlled dismantling and
engulfment of damaged or stressed cells. The Bcl-2 protein family regulates the intrinsic
apoptotic pathway that leads to mitochondrial outer membrane permeabilisation (MOMP).
Proteins in this family are defined by the presence of Bcl-2 homology (BH) domains and
function either in a pro-apoptotic or pro-survival manner. Pro-survival family members
sequester pro-apoptotic relatives by binding the amphipathic BH3 α-helix of the proapoptotic protein within a hydrophobic surface groove on the pro-survival protein. BH3
helices also play important roles in activation of critical pathway effectors Bax and Bak, both
of which oligomerise on the mitochondrial outer membrane (MOM) to permeabilise it,
releasing cytochrome c which initiates apoptosis1. Proteins of another subfamily, called BH3only proteins, are directly and/or indirectly responsible for Bax and Bak activation2.
Bok is a third potential effector protein that shares sequence homology with Bax and Bak and
induces apoptosis when overexpressed3. It was originally thought to function by
permeabilising the MOM like Bax and Bak4. However, recent data suggest it cannot
permeabilise the MOM in the absence of Bax and Bak4. The structure and function of Bok
has remained elusive despite the 16 years of investigation since its discovery, due in part to
the lack of a suitable recombinant expression system. Producing Bok recombinantly could
lead to elucidation of its structure, its role in inter-family interactions and any wider roles
Bok may have within cells.
References
[1] Youle R.J. and Strasser A. (2008) “The BCL-2 protein family: opposing activities that mediate
cell death” Nat. Rev. Mol. Cell Biol. 9:47–59.
[2] Kuwana T., Bouchier-Hayes L., Chipuk J.E., Bonzon C., Sullivan B.A., Green D.R. and
Newmeyer D.D. (2005) “BH3 Domains of BH3-only proteins differentially regulate Baxmediated mitochondrial membrane permeabilization both directly and indirectly” Mol. Cell
17:525–535.
[3] Hsu S.Y., Kaipia A., McGee E., Lomeli M., and Hsueh A.J. (1997) “Bok is a pro-apoptotic Bcl-2
protein with restricted expression in reproductive tissues and heterodimerizes with selective antiapoptotic Bcl-2 family members” Proc Natl Acad Sci USA 94:12401–12406.
[4] Echeverry N., Bachmann D., Ke F., Strasser A., Simon H.U. and Kaufmann T. (2013)
“Intracellular localization of the BCL-2 family member BOK and functional implications” Cell
Death Differ.
73
Implications of the cysteine-tyrosine crosslink in cysteine
dioxygenase
M. Fellner1, R.J. Souness1, E.P. Tchesnokov1, S.M. Wilbanks2 and G.N.L. Jameson1
1
Department of Chemistry, University of Otago, Dunedin 9054, New Zealand
Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand
2
The enzyme cysteine dioxygenase (CDO) oxidises cysteine to its sulfinic acid, the first step in
oxidative breakdown of cysteine. Due to its role in regulating cysteine levels it is linked to
various diseases including cancer, rheumatoid arthritis and several neurological diseases,
such as Alzheimer's and Parkinson's diseases. Crystal structures of mammalian CDO [1-4]
show a ferrous ion in the active site coordinated by three histidines and in close proximity to
an unusual posttranslational modification, a covalent cysteine-tyrosine crosslink between C93
and Y157. Although the cysteine and tyrosine which form the crosslink are completely
conserved in eukaryotes, sequence alignment of putative CDOs from prokaryotes shows that
only the tyrosine is conserved and the cysteine is often replaced by a glycine at the
corresponding position [5].
Here we present a crystallographic investigation of this crosslink by comparing the
eukaryotic CDO from Rattus norvegicus with Pseudomonas aeruginosa CDO, a prokaryotic
homologue that lacks the crosslink. Distinct crystallization conditions yielded a number of
new crystal structures with interesting features. The new crystal structures open up interesting
insights into the effect of crystallization methods used to obtain the structures. Combined
with enzymatic assays, these structural studies probe how the active site cofactor and residues
contribute to catalysis of the dioxygenation of thiols. These insights suggest possible
implications for the biological background of non-heme, mono-iron enzymes.
References
[1] Ye S., et al., (2007), "An insight into the mechanism of human cysteine dioxygenase. key roles of the thioether-bonded
tyrosine-cysteine cofactor", J. Biol. Chem., 282: 3391-3402.
[2] Simmons C.R., Hao Q. and Stipanuk M.H. (2005), "Preparation, crystallization and x-ray diffraction analysis to 1.5 Å
resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation", Acta
Crystallogr., Sect. F Struct. Biol. Cryst. Commun., F61: 1013-1016.
[3] Souness R.J., et al. (2013), "Mechanistic implications of persulfenate and persulfide binding in the active site of cysteine
dioxygenase" Biochemistry, 52: 7606-17.
[4] Driggers C.M., et al. (2013), "Cysteine dioxygenase structures from pH4 to 9: consistent cys-persulfenate formation at
intermediate pH and a Cys-bound enzyme at higher pH", J Mol Biol, 425: 3121-36.
[5] Dominy J.E., Jr., et al. (2006), "Identification and characterization of bacterial cysteine dioxygenases: a new route of
cysteine degradation for eubacteria", J. Bacteriol., 188: 5561-5569.
74
Into the future with CIF: advances in data, metadata and metametadata applications
Nick Spadaccini, Doug du Boulay and Syd Hall
School of Chemistry and Biochemistry, University of Western Australia, Crawley 6009, Australia.
When CIF was adopted as an information exchange standard by the IUCr in 1991, it was, if
anything, ahead of the then state of the art. Free-format, extensible, low-overhead - it offered
an easy route to implementation by scientific software. By externalizing the semantics of its
tags to external dictionaries, it allowed ontology development to be decoupled from format,
and thus had an important role to play in interoperability between purely crystallographic
applications and the wider worlds of structural biology, chemical informatics and laboratory
data management [1].
The latest enhancements to CIF [2-4], with the introduction of a more powerful dictionary
definition language DDLm that support methods definitions (in dREL) and multiple data
models, provide unrivalled potential for taking computer ontologies into completely new
territories. This paper will demonstrate the latest Java-based CIF browser, and the increased
flexibility of CIF2 in data deposition, validation and extension.
References
[1]
[2]
[3]
[4]
Hall S.R. and McMahon B. (Eds) “Definition and exchange of crystallographic data” International Tables For
Crystallography, Volume G., I, Springer, (2005).
Spadaccini N. and Hall S.R. (2012) “Extensions to the STAR file syntax.” J Chem. Inf. Model., 52:1901-6.
Spadaccini N. and Hall, S.R. (2012) “DDLm: A new dictionary definition language.” J Chem. Inf. Model., 52:190716.
Spadaccini N., Castleden I. R., du Boulay D. and Hall, S.R. (2012) “dREL relational expression language for
dictionary methods” J Chem. Inf. Model., 52:1917-25.
75
Molecular basis of signaling by Toll/Interleukin-1 Receptor domaincontaining adaptors in Toll-like Receptor pathways
Shane Horsefield1, Thomas Ve1, Bostjan Kobe1.
1
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia
The innate immune system is a complex system for protecting an organism from harmful
agents causing disease. Recognition of pathogenic organisms is an integral role of this system
and involves an essential set of receptors, Toll-like receptors (TLRs). These TLRs are
pattern-recognition receptors (PRRs) and recognise pathogen-associated molecular patterns
(PAMPs) [1]. To date, there are 10 human TLRs and together, recognise a range of PAMPs,
such as flagellin, lipopeptides, uropathogenic bacteria, lipopolysaccharide (LPS), DNA, RNA
and CpG-DNA [2].
Initiation of the immune response begins with PAMP binding to the extracellular domain of
the TLR and sends a signal to the intracellular domain called the Toll/interleukin-1 receptor
(TIR) domain. This TIR domain is central to TLR signaling and is also present in
downstream adaptor molecules such as MyD88 (myeloid differentiation primary response
gene 88), MAL, (MyD88 adapter-like or TIRAP), TRIF (TIR domain-containing adapter
inducing IFNβ) and TRAM (TRIF- related adapter molecule). . The TIR domain of TLRs
interacts with the TIR domain of adaptor molecules to continue the signaling cascade to
induce the production of pro-inflammatory cytokines and type-1-interferon (type-1-IFN)
responses [3,4].
The ability of the adaptors to activate distinct TLR signaling pathways makes them attractive
candidates for drug development, and a detailed structural understanding of receptor-adaptor
and adaptor-adaptor TIR domain complexes including their stoichiometry and order of
assembly is therefore needed. We have previously solved the crystal structure of the MAL
TIR domain to 3.0 Å resolution, and the overall fold displayed significant structural
differences compared to other TIR domain structures [5] We are now characterizing the
interaction between MAL and MyD88, and the receptors; TLR4 and TLR5 in more detail,
using crystallography and site directed mutagenesis in combination with pull-down/coimmunoprecipitation assays, and biophysical methods such as microscale thermophoresis,
and bio-layer interferometry.
References
[1] Kawai T., and Akira S. (2010) “The role of pattern-recognition receptors in innate immunity: update on Toll-like
receptors” Nature Immunology 11: 373-384.
[2] Gay,N.J., and Gangloff M. (2007) “Structure and function of Toll receptors and their ligands” Annual Review of
Biochemistry 76: 141-165.
[3] O'Neill L.A., Golenbock D. and Bowie A.G. (2013) “The history of Toll-like receptors - redefining innate immunity”
Nature Reviews. Immunology 13, 453-460.
[4] Ve T., Gay N.J., Mansell A., Kobe B. and Kellie S. (2012) “Adaptors in toll-like receptor signaling and their potential
as therapeutic targets” Current Drug Targets 13: 1360-1374.
[5] Valkov E., Stamp A., Dimaio F., Baker D., Verstak B., Roversi P., Kellie S., Sweet M.J., Mansell A., Gay N.J., et al.
(2011) “Crystal structure of Toll-like receptor adaptor MAL/TIRAP reveals the molecular basis for signal transduction
and disease protection” Proceedings of the National Academy of Sciences of the United States of America 108: 1487914884.
76
Neutron study of the magnetic structures and phase transition in
iron (III) phosphate, FePO4
Christopher J. Howard1, Paul J. Saines2, James R. Hester3 and Anthony K Cheetham,4
1
School of Engineering, University of Newcastle, Callaghan, NSW 2308, Australia.
Department of Chemistry, University of Oxford, Oxford OX1 3QR, UK.
3
Bragg Institute, Australian Nuclear Science and Technology Organisation, Private Mail Bag 1,
Menai, NSW 2234, Australia.
4
Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage
Road, Cambridge CB3 0ES, UK.
2
Neutron powder diffraction has been used to re-investigate the magnetic structures and phase
transitions in anhydrous iron (III) phosphate, FePO4. The compound crystallizes in space
group P3121 and is paramagnetic at room temperature. Below 24 K it becomes
antiferromagnetic with moments lying in the basal plane, and below a spin orientation
transition at 17 K the moments turn to be perpendicular to the basal plane [1]. The data
analysis was preceded and informed by an examination of possible magnetic symmetries
using the group theory as implemented in computer program ISOTROPY [2]. The
refinements were carried out using GSAS [3], making use of the magnetic symmetries
defined by ISOTROPY.
The magnetic structures are characterized by cell doubling relative to the chemical structure,
and are therefore associated with distortions (irreps) at the A-point (k = 0,0,1/2) of the
Brillouin zone. The magnetic space group for the lower temperature structure is evidently
Pc3221 (#154.44). The BNS Wyckoff position 6b [2,4] permits an antiferromagnetic
arrangement of Fe3+ moments, with components both along the three-fold axis and in the
basal plane. It has not been possible to refine both components, but the good fit obtained
assuming an axial component only suggests that the basal plane component might be
negligible. Above the spin reorientation transition, the magnetic space group indicated by
ISOTROPY is Cc2 (#5.16). Note the loss of three-fold symmetry. The Fe3+ are now
distributed over BNS Wyckoff positions 4a and 8c. An antiferromagnetic arrangement of
moments results, with those on 4a being symmetry constrained to lie along the y-axis, and
those on 8c not so constrained. Although GSAS does not work directly with BNS Wyckoff
positions, it is generally possible to describe the situation using normal Wyckoff positions
with moments suitably constrained – e.g. 4a in #5.16 can be described using 2a and 2b in #5.
The ISOTROPY analysis, including the setting of the magnetic structures for refinement
using GSAS, appears in the ISOTROPY tutorial [2] as Case Study 4.
The data were recorded at 4, 8, 11, 14, 16, 18, 20, 22, 24, 27 K and room temperature using
powder diffractometer ECHIDNA at the Opal research reactor. The structures are indeed
consistent with those reported previously [1], and the temperature evolution of the Fe3+
magnetic moment (determined from the refinements) can be followed in detail.
References
[1] Battle P.D., Cheetham A.K., Gleitzer C., Harrison W.T.A., Long G.J. and Longworth G. (1982), “A novel magnetic
phase transition in anhydrous iron (III) phosphate, FePO4”, J. Phys.C: Solid State Phys., 15, L919-924.
[2] ISOTROPY software suite, iso.byu.edu.
[3] Larson A.C. and Von Dreele R.B. (2004), “General Structure Analysis System (GSAS)”,Los Alamos National
Laboratory Report LAUR 86-748.
[4] Gallego S.V., Tasci E.S., de la Flor G., Perez-Mato J.M. and Aroyo M.I. (2012), "Magnetic symmetry in the Bilbao
Crystallographic Server: a computer program to provide systematic absences of magnetic neutron diffraction" J. Appl.
Cryst. 45 1236-1247.
77
Structural characterization of the mammalian CAD multienzyme
complex
Yujung Jeon1, Ian L. Ross2, Michael J. Landsberg2, Ben Hankamer2, and Bostjan Kobe1
1
School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia,
Queensland, Australia
2
Institute for Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia
CAD is a large, tri-functional multi-enzyme complex (~250 kDa), which is comprised of
three covalently linked domains:
carbamoyl-phosphate synthase (CPS), aspartate
transcarbamylase (ATC) and dihydroorotase (DHO). With the length of over 2000 amino
acids, it is one of the largest known polypeptides, and this oligomeric protein is known to be
involved in the initial stage of de novo pyrimidine biosynthesis required for the pyrimidine
nucleotides and cell growth.
In prokaryotes, individual domains exist as separate proteins. While some structural
information exists on these bacterial proteins and their association, little is known about the
assembly of the mammalian CAD complex. The complex has been proposed to form a homohexamer of ~1.5MDa.
For the structural characterization, two strategies will be pursued in parallel time: 1. Single
particle cryo-electron microscopy, to obtain a low resolution structure of the complex, and 2.
Crystallography followed by the crystallization of either the complex or individual domains
for better resolution structure. The combination of the two approaches would enable us to
obtain a pseudo-high-resolution model.
Different from the previous studies of the individual domains of the protein, this project will
be focused on the production of the whole CAD complex in an assembled and functional
state. Whereas it is known that over-expression of CAD is usually associated with the
proliferating and tumour cell development, the understandings of the structural basis of the
complex will potentially help developing anti-tumour drugs.
78
The functional and structural role of cysteine residues in Toll-like
receptor signalling adaptor MAL/ TIRAP
Peter Lavrencic1,2,4, Thomas Ve1,2,4, Mehdi Mobli1,2,3, Bostjan Kobe1,2,4
1
School of Chemistry and Molecular Biosciences, The University of Queensland, QLD, 4072,
Australia
2
Institute for molecular Biosciences, The University of Queensland, QLD, 4072, Australia
3
Centre for Advanced Imaging, The University of Queensland, QLD, 4072, Australia
4
Australian Infections Disease Research Centre, The University of Queensland, QLD, 4072, Australia
MyD88 adaptor protein (MAL) / TIR domain-containing adaptor protein (TIRAP) is a key
player in the Toll-like receptor (TLR) signalling cascade of the human immune system. As
the TLRs are stimulated, the Toll/ interleukin-1 receptor (TIR) domain of MAL binds to the
intracellular domain of the TLR to relay the signal to MyD88 and in turn activate the
production of NF-κB and cytokines.
Our group recently solved the crystal structure of MAL, which showed that specific cysteines
formed two disulphide bonds that could contribute to the stability of the protein[1]. Following
NMR sequential protein backbone assignment, data revealed that the protein contained four
reduced cysteine residues in solution. In addition, another two cysteine residues appear to be
reactive and may be involved in redox-regulated binding and signal transduction. Future
work will be focused on the functional role of cysteines in the MAL protein.
TIR domains have been shown to become active when they form dimers. A stable dimer of
the MAL TIR-domain has been purified and confirmed using both small angle laser
scattering and multi angle light scattering. Future work will focus on crystallizing the MAL
TIR domain dimer to reveal the interacting interface and the possible functional significance
of the dimer form.
References
[1]
Valkov E. et. al. (2011) Crystal structure of Toll-like receptor adaptor MAL/TIRAP reveals the molecular basis for
signal transduction and disease protection, Proc. Natl. Acad. Sci. USA., 108(36), 14879-14884.
79
Structurally guided small molecule targeting of the insulin and
Type 1 insulin-like growth factor receptors
C.F. Lawrence1,2, J.G. Menting1,2, M.B. Margetts1,2, K.E. Jarmin1,2, K.N. Lowes1,2,
G. Lessene1,2, K. Lakovic1,2 and M.C. Lawrence1,2
1
Walter and Eliza Hall Institute of Medical Research, 1G royal Parade, Parkville, Australia.
Department of Structural Biology, Faculty of Medical Biology, University of Melbourne, Parkville,
Victoria, 3052, Australia.
2
The insulin receptor (IR) and Type 1 insulin-like growth factor receptor (IGF-1R) are
homodimeric α2β2 glycoproteins that together, with the insulin-receptor related receptor,
form the insulin receptor family of receptor tyrosine kinases. While both IR and IGF-1R
possess high-affinities for their cognate ligands (insulin, and IGF-I and IGF-II, respectively),
they can also bind and be activated by the reciprocal ligands. Activation of either receptor
affects downstream PI3K- AKT and RAS-RAF-MAPK signalling, with varying effects on
cellular glucose metabolism, differentiation and proliferation. Aberrant signalling leads to a
number of clinical manifestations including diabetes (in the case of IR) and cancer (in the
case of IGF-1R and possibly IR), making both receptors attractive pharmaceutical targets.
The primary extracellular ligand binding site of either receptor is a tandem element
comprising the first leucine-rich repeat domain (L1) of one receptor a-chain, in association
with the C-terminal region (αCT) of the opposite receptor α-chain. Disruption of the L1 and
αCT association disrupts ligand binding. 114,000 small-molecules within an in-house library
were screened for their ability to disrupt this site within the IGF-1R system. Identified hits
were subsequently confirmed in an equivalent surface plasmon resonance assay. The
agonistic or antagonistic potential of the identified compounds as well as their binding
kinetics are being determined and further refinement of hit compounds will require X-ray
crystallographic and computational pharmacophore determination, of which, recent progress
has been made.
80
Structural characterisation of the retromer complex and associated
sorting nexins
Natalya Leneva1, Suzanne Norwood1, Rajesh Ghai1, Nathan Cowieson2, Anthony Duff 3,
Kathleen Wood3 and Brett Collins1
1
Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072.
Australian Synchrotron, 800 Blackburn Road, Clayton, VIC, 3168.
3
Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW,
2232.
2
Retromer is a peripheral membrane protein complex that plays a critical role in a broad range
of physiological, developmental and pathological processes including Wnt signalling, toxin
transport and amyloid production in Alzheimer’s disease.
The classical mammalian retromer complex consists of a core heterotrimeric cargo
recognition sub-complex (VPS26, VPS29 and VPS35) associated with a dimer of proteins
from the SNX–BAR sorting nexin family (SNX1/SNX2 and SNX5/SNX6) that drives
membrane deformation and tubulation. By recruiting the cargo-selective sub-complex to the
forming tubules, the SNX–BAR coat complex mediates the retrograde transport of proteins
from endosomes to the trans-Golgi network.
Recent studies have highlighted the molecular and functional diversity of retromer and the
identification of new interacting proteins, including the WASH complex, has revealed that
the role of retromer extends to aspects of endosome-to-plasma membrane sorting and
regulation of signalling events. Emerging evidence indicates that cargo specificity is
mediated by specific sorting nexins. These include SNX3, involved in the trafficking of the
Wntless/MIG-14 protein, and SNX27, a PX-FERM protein that mediates the retrieval of the
β2-adrenergic receptor.
Using the SAXS/WAXS and MX beamlines at the Australian Synchrotron, we have acquired
crystallographic and small angle scattering data to determine how the core cargo recognition
sub-complex assembles and to characterise the retromer-associated sorting nexins.
Intriguingly, the core trimeric complex is able to form a symmetric dimer, which may have
implications for functional interactions in vivo. We are using this structural information in
combination with biochemical and biological studies in a synergistic approach to understand
retromer-mediated endosomal protein sorting and how this fascinating protein complex
contributes to a diverse set of cellular processes.
The retromer complex is conserved across all eukaryotes. We are also currently exploring the
structure of these proteins in the thermophilic fungus Chaetomium thermophilum and initial
crystallisation experiments have produced some promising results.
81
Structural elucidation of zinc acquisition mechanisms in
Streptococcus pneumoniae and development of novel antibiotics
against streptococcal diseases
Zhenyao Luo1, Rafael Couñago1, Alastair McEwan1, Christopher McDevitt2 and Boštjan
Kobe1
1
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland,
Australia
2
Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of
Adelaide, Adelaide, Australia
Streptococcus pneumoniae is among the world's leading bacterial pathogens and is
responsible for over 1 million deaths annually [1]. Transition metals, exemplified by Zn2+,
have been recognized as essential nutrients for the growth and pathogenicity of pathogenic
bacteria in human [2]. Acquisition of Zn2+ in S. pneumoniae is supported by ATP binding
cassette transporter AdcBC, including two surface proteins AdcA and AdcAII as the initial
receptors for Zn2+ recognition [3]. AdcA and AdcAII are high affinity Zn2+-binding proteins
(BP) that belong to Cluster A-I substrate binding protein (SBP) subgroup [4] and their
imperative roles in S. pneumoniae survival and virulence make them potential therapeutic
targets for the development of novel antimicrobial drugs against streptococcal diseases [5].
However, the lack of experimentally determined structures of AdcA and apo state of AdcAII
hinders deeper understanding towards SBP-mediated Zn2+ binding and transport mechanisms
at molecular level. Therefore, in this study, we aim to determine the molecular structures of
AdcA (Zn2+-bound and apo) and AdcAII (apo) by X-ray crystallography and identify
potential AdcA and AdcAII inhibitors by high throughput screening. The outcome of this
research will provide the first structural insights into the mechanism of AdcA and AdcAIIfacilitated acquisition of Zn2+ and offer molecular blueprints for rational design and
development of novel AdcA and AdcAII inhibitors against streptococcal diseases.
References
[1] Pneumococcal vaccines : WHO position paper. 1999.
[2] Klein, J.S. and O. Lewinson (2011), “Bacterial ATP-driven transporters of transition metals: physiological roles,
mechanisms of action, and roles in bacterial virulence”, Metallomics 3(11):1098-108.
[3] Bayle, L., et al. (2011), “Zinc uptake by Streptococcus pneumoniae depends on both AdcA and AdcAII and is essential
for normal bacterial morphology and virulence”, Mol Microbiol, 82(4): 904-16.
[4] Berntsson, R.P., et al..010), “A structural classification of substrate-binding proteins”., FEBS Lett, 584(12): 2606-17.
[5] Counago, R.M., et al. (2012), “Prokaryotic substrate-binding proteins as targets for antimicrobial therapies”, Curr Drug
Targets 13(11): 1400-10.
82
Phylogenetic mapping and structural comparison of bacterial ketolacid reductoismeraes
You Lv, Luke W. Guddat, Bostjan Kobe and Mark A. Schembri
School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre,
University of Queensland, St Lucia, Queensland, Australia
Urinary tract infections (UTIs) are one of the most common bacterial infections of humans.
UTIs also represent a significant burden on healthcare systems, a problem exasperated by
increasing levels of antibiotic resistance. Uropathogenic Escherichia coli (UPEC) are the
leading cause of UTI, however the infection is also caused by many other Gram-negative and
Gram-positive bacterial pathogens. We recently showed that the genes involved in the
production of branched chain amino acids leucine, isoleucine and valine are required for
UPEC growth in urine. As the biosynthetic pathway for the synthesis of these amino acids is
absent in humans, inhibition of this pathway may therefore represent a novel approach to
treating UTI. In this study we have focused on the ilvC gene, encoding ketol-acid
reductoisomerase (KARI; EC 1.1.1.86), as a putative novel target for preventing UTI. We
performed a comprehensive phylogenetic analysis of bacterial KARIs and showed that the
sequences cluster into several specific groups. The crystal structure of KARI from E. coli
and P. aeruginosa have been determined, and we have performed a comparative analysis of
these two enzyme. We currently examining the structure of KARI from several other
uropathogens in combination with mutagenesis approaches to confirm the general
requirement for branched chain amino acid biosythesis by all uropathogens. Our study may
lead to the development of a novel class of drugs to treat UTI.
83
Single molecule magnetism in µ-phenolato dinuclear lanthanide
complexes containing heptadentate Schiff base ligand
Melina Nematirad1, Stuart R. Batten and Keith S. Murray
1
School of Chemistry, University of Monash, clayton, 3800, VIC, Australia.
The heptadentate Schiff base ligands, 2-(2-hydroxyphenyl)-1,3-bis[4-(2-hydroxyphenyl)-3azabut-3-enyl]-1,3-imidazoline (H3api) [1], the 3-pyridyl substituted derivative H3hep, and
three more derivatives with 3-cyanophenyl (H3cic), 4-cyanophenyl (H3zci) and 4methoxyphenyl (H3mpt) substituents yield [Lnapi]2 [2], [Lnhep]2 , [Lncic]2 , [Lnzci]2 and
[Lnmpt]2 species when combined with lanthanide salts under basic conditions. A survey of
the magnetic properties of these dinuclear lanthanide complexes (Ln = Gd, Tb, Dy, Ho) has
identified single molecule magnetic behavior in the cases of [Dyapi]2 [3] and [Dyhep]2.
Figure 1: Tripodal ligands H3api, H3hep, H3cic, H3zci and H3mpt and the crystal structure of
the [Dyhep]2 complex.
References
[1]
Howell R., Spence K., Kahwa I., and Williams D (1998) “Structure and luminescence of the neutral dinuclear
lanthanide(III) complexes [{Ln(api)}2] {H3api = 2-(2-hydroxyphenyl)-1,3-bis[4-(2-hydroxyphenyl)-3-azabut-3-enyl]1,3-imidazolidine}’’J. Chem. Soc., Dalton Trans, 2727-2734.
[2] Isobe T., Kida S. and Misumi S (1967) “ Preparation of the Schiff base complexes of lanthanide (III) ions’’, Bull.
Chem. Soc. Jpn, 40:1862-1863.
[3] Nematirad M., Gee W., Langley S., Chilton N., Moubaraki B., Murray K., Batten R. (2012) “Single molecule magnetism
in a µ-phenolato dinuclear lanthanide motif ligated by heptadentate Schiff base ligands’’, Dalton Trans, 41:1371113715.
84
Explicit and implicit data merging in crystallographic least squares
A. David Rae
Research School of Chemistry, The Australian National University, Canberra, ACT 0200, Australia
E-mail: rae@rsc,anu,edu.au
The model for the diffraction pattern of a crystal structure can be described in various ways
We start with the segmentation of intensities at points in reciprocal space into uncorrelated
contributions from the background and fractions of coexisting integrated intensities and end
up with a structure model defining the integrated intensities and those additional parameters
used to evaluate the multiplying factors needed to best model the observed diffraction pattern.
Information about the various parameters is duplicated to various amounts in different parts
of the diffraction data.
A recent review article [1] showed how an intensity |Y(h)|2 associated with a sampling point
in reciprocal space can be restructured in terms of partial observations Yn(h)2 = fn(h)|Y(h)|2
that describe uncorrelated components of |Y(h)|2. Thus Y(h) is the vector sum of orthogonal
components Yn(h) in and the direction of Y(h) is defined by an initial model that evolves with
refinement. For any least squares refinement cycle the direction and magnitude of the
observed value of Y(h) is assumed to be fixed but does have a variance. The variance of a
partial residual Δhn = Yn(h)obs - Yn(h)calc can also be subdivided into contributions. Thus we
can assess the fraction of a residual that is being refined and how much of that fraction is
associated with individual parameters or combination of parameters. If we assume a variance
for an unrefined parameter or combination of parameters we can evaluate what fraction of the
total number of observations this is associated with. These ideas avoid the maximum
ignorance concept that there is only a single global model for error distribution.
In least squares proper notice should be taken of the duplication of partial observations. If we
say that Δj = (Yobs)j – (Ycalc)j and all the (Ycalc)j = Ycalc then Σj wj Δj2 = w Δ2 where w = Σj wj and
Δ2 = [<Yobs> - Ycalc]2 + [<Yobs2> - <Yobs>2]
where <Yobs> = Σj fj (Yobs)j <Yobs2> = Σj fj (Yobs)j2 fj = wj /w and Σj fj = 1. We see that the
variance of duplicates of a partial observation can be separated from the variance of the
model of a partial observations and the same weighting is used to obtain both the averages
<Yobs2> and <Yobs>.
These ideas can be developed to define the effective number of observations associated with
components of a diffraction pattern and the variances of the integrated intensities and
backgrounds used to describe it.
References
[1] Rae. A.D. (2013) “The use of partial observations, partial models, and partial residuals to improve the least squares
refinement of crystal structures ”, Crystallography Reviews, 20: 155-229.
85
Elaboration of benzoylurea inhibitors targeting pro-survival Bcl-xL
Michael Roy1,2,Amelia Vom1,2, Soo San Wan1, Hong Yang1, Brian Smith3, Peter Colman1,2,
Guillaume Lessene1,2,4 and Peter Czabotar1,2
1
Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.
Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia.
3
La Trobe Institute for Molecular Sciences, La Trobe University, Bundoora, Victoria, Australia.
4
Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, Victoria,
Australia.
2
Interactions between members of the Bcl-2 family of proteins control the life/death fate of
cells by regulating apoptosis. Cancer cells can evade apoptosis through over-expression of
pro-survival members of the Bcl-2 family, such as the proteins Bcl-xL and Mcl-1. This is not
only an important step in the progression to cancer but also a mechanism through which
cancer cells can become resistant to standard anti-cancer therapies1,2,3.
Small molecules able to mimic the activity of pro-apoptotic BH3-only proteins hold potential
for reactivating apoptosis in tumours; either as single agents in certain tumours, or to sensitise
cancers to existing therapies4,5. Previous work at WEHI has led to the development of small
molecules possessing a benzoylurea core which are able to mimic alpha-helical BH3 peptides
and which bind to Bcl-xL with low micromolar binding affinity6. Co-crystal structures for a
number of these compounds in complex with Bcl-xL have been obtained and have formed the
basis for further structure-based optimization.
This presentation will outline new insights gained during this ongoing structure-guided
medicinal chemistry campaign, including discovery of a novel 300 nM inhibitor of prosurvival Bcl-xL.
References
[1] Adams J.M. and Cory S. (2007)“The Bcl-2 apoptotic switch in cancer development and therapy”, Oncogene, 26:1324–
1337.
[2] Amundson S.A. et al.(2000) “An informatics approach identifying markers of chemosensitivity in human cancer cell
[3]
[4]
[5]
[6]
lines” Cancer Research 60:6101–6110.
Kelly P.N. and Strasser A. (2011)“The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer
therapy” Cell Death and Differentiation 18:1414–1424.
Roy M.J. et al. (2013)“Cell death and the mitochondria: therapeutic targeting of the BCL-2 family-driven pathway”
British Journal of Pharmacology [30 Sep 2013, Epub ahead of print]
Lessene G., Czabotar P.E. and Colman P.M.(2008) “BCL-2 family antagonists for cancer therapy” Nature Reviews.
Drug Discovery 7:989–1000.
Brady R.M. et al. (2014) “De-Novo designed library of benzoylureas as inhibitors of BCL-XL: synthesis, structural and
biochemical characterization” Journal of Medicinal Chemistry [23 Jan 2014, Epub ahead of print].
86
Crystallization and structure characterization of the Gun4 from
Chlamydomonas reinhardtii
ShabnamTarahi Tabrizi1, Anthony Duff 2, Stephen J. Harrop3, Arthur Sawicki1, Robert D.
Willows1
1
Department of Chemistry and Biomolecular Sciences, Macquarie University, New South Wales, 2109
Australia 2ANSTO, National Deuteration Facility Lucas Heights, 3 School of Physics, Faculty of
Science, University of New South Wales
The GENOMES UNCOUPLED4 (GUN4) protein is a regulatory subunit of Mg-chelatase,
the enzyme that inserts magnesium into protoporphyrin IX, in the chlorophyll biosynthesis
pathway. GUN4 binds the chlorophyll biosynthesis intermediates, protoporphyrin and Mg
protoporphyrin, stimulates Mg chelatase activity, and is implicated in developmental
signaling pathway between the chloroplast and nucleus. His-tagged GUN4 expressed in
Escherichia coli has been crystallized by the vapour-diffusion method using 1M Ammonium
citrate tribasic at pH 7.0 as a precipitant. Structure determination will shed more light on the
structure and function of GUN4 in the chlorophyll biosynthesis pathway. Also, comparison of
the crystal structure with the low resolution solution structure (SAXS) of the eukaryotic
GUN4 provides a model for the structure of the extra C-terminal domain of GUN4 not
present in structurally determined homologues. The extra domain, which is phosphorylated
and is required for magnesium chelatase activity, is located at the end of the main molecular
axis.
References
[1] Larkin R.M., Alonso J.M., Ecker J.R. and Chory J. (2003) “GUN4, a regulator of chlorophyll synthesis and intracellular
signaling”, Science 299:902-906.
[2] Davison, P.A., Schubert H.L., Reid J.D., Iorg C.D., Heroux A., Hill C.P., and Hunter C.N. (2005) “Structural and
biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis”, Biochemistry
44:10.
[3] Willows R.D., Hansson M., Beale S.I., Laurbergd M., and Al-Karadaghi S. (1998) “Crystallization and preliminary Xray analysis of the Rhodobacter capsulatus magnesium chelatase BchI subunit”, Biological Crystallography D55:2.
87
Multiple binding modes of isothiocyanate inhibitors of macrophage
migration inhibitory factor define structure activity relationships
Joel D.A. Tyndall,1 Emma S. Spencer,2 Edward J. Dale,3Aimée L. Gommans,4
Malcolm T. Rutledge,4Christine T. Vo,1Yoshio Nakatani,4 Allan B. Gamble,1
Robin A. J. Smith,3 Sigurd M. Wilbanks,4and Mark B. Hampton2
1
National School of Pharmacy,University of Otago, Dunedin9054, New Zealand
Centre for Free Radical Research, Department of Pathology, University of Otago, Christchurch
8140, New Zealand
3
Department of Chemistry, University of Otago, Dunedin, 9054, New Zealand
4
Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, 9054,
New Zealand
2
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with roles in
inflammatory response and is elevated in diseases such as arthritis, sepsis and cancer. The
biological activity of MIF is triggered via binding to the cell surface receptor CD74 as well as
intracellular signaling proteins. MIF also possesses tautomerase activity catalyzed via the Nterminal proline and efforts are being made to develop small molecule inhibitors for clinical
use. The biological substrate for this enzymatic activity has as yet been identified, however
inhibition at this site antagonizes the interaction with CD74. Isothiocyanates (ITCs) are
phytochemicals found in Brassicavegetables and have been identified as potent irreversible
inhibitors of MIF.1,2 With phenethyl isothiocyanate (PEITC; IC50 2 µM)1being the first
compound tested, at least 25 ITCs with structural similarity to PEITC have been tested in
order to identify structure-activity relationships and a lead compound for in vivo testing.
Crystallographic analysis of five ITC-MIF complexes has revealed some unusual and
unexpected findings when compared to our initial PEITC-MIF complex.2 Multiple structures
of benzyl isothiocyanate (BITC) complexed with the trimeric MIF revealed different binding
modes between active sites within one trimer, which then differed from a separate BITC-MIF
complex that revealed a single but highly strained orientation. These results have ultimately
allowed us to simplify a SAR in our search to identify more potent ITC inhibitors of MIF.
References
[1] Brown K.K., Blaikie F.H., Smith R.A., Tyndall J.D.A., Lue H., Bernhagen J., Winterbourn C.C. and Hampton M.B.
(2009) “Direct modification of the proinflammatory cytokine macrophage migration inhibitory factor by dietary
isothiocyanates.” J. Biol. Chem 284:32425-32433
[2] Tyndall J.D., Lue H., Rutledge M.T., Bernhagen J., Hampton M.B. and Wilbanks S.M. (2012) “Macrophage migration
inhibitory factor covalently complexed with phenethyl isothiocyanate.”Acta Crystallogr Sect F Struct Biol Cryst
Commun 68(Pt 9):999-1002
88
A novel N-terminal domain may dictate the role of the disulfide
bond protein α-DsbA2 from Wolbachia pipientis
Patricia M Walden1, Premkumar Lakshmanane1, Fabian B Kurth1, Iñaki Iturbe-Ormaetxe2,
Maria A Halili1, Julia K Archbold1, Begoña Heras3 and Jennifer L Martin1
1
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Queensland, 4072,
Australia
2
Monash University, Faculty of Science, Melbourne, Victoria, 3800, Australia
3
Department of Biochemistry, La Trobe University, Melbourne, Victoria, 3086, Australia
Wolbachia pipientis, a bacterium from the family of Rickettsiacaeae, infects around 65% of
existing insect species [1]. Importantly, Wolbachia can infect Aedes aegypti, a mosquito that
transmits the dengue virus, and inhibits the replication of dengue within the mosquito [2].
However, the Wolbachia-host interaction is poorly understood. We hypothesize that
Wolbachia secretes proteins through its type IV secretion system to influence host
physiology. Like many other bacteria, Wolbachia holds disulfide bond forming (Dsb)
proteins that introduce disulfide bonds into secreted effector proteins. The genome of the
Wolbachia strain wMel encodes two DsbA-like proteins, alpha-DsbA1 and alpha-DsbA2, and
an integral membrane protein, alpha-DsbB. Alpha-DsbA1 and alpha-DsbA2 both have a CysX-X-Cys active site that, by analogy with Escherichia coli DsbA, would need to be oxidized
to the disulfide form to serve as a disulfide bond donor toward substrate proteins. We
previously showed that the integral membrane protein alpha-DsbB oxidizes alpha-DsbA1, but
not alpha-DsbA2 [3].
Here we describe the structural and biochemical characterization of alpha-DsbA2. This
protein has a novel N-terminal extension, predicted to be a transmembrane domain, that is not
present in any of the bacterial DsbA homologues studied to date. Crystallization of the fulllength protein containing this N-terminal domain proved difficult, however the structure of
the catalytic domain was solved by MR using remote sequence homologs. We carried out a
series of biochemical assays with the full-length and catalytic alpha-DsbA2 constructs and
identified that the function of alpha-DsbA2 is dictated by its N-terminal domain. This is the
first extensive investigation of alpha-DsbA2 from Wolbachia revealing that this protein has a
novel role in the Wolbachia Dsb folding machinery. Exploiting Wolbachia for halting the
spread of Dengue fever is an ongoing strategy in tropical climates.
References:
[1]
[2]
[3]
Hilgenboecker K., Hammerstein P., Schlattmann P., Telschow A. and Werren J.H. (2008) “How many species are
infected with Wolbachia?--A statistical analysis of current data”, FEMS Microbiol Lett 281: 215-220.
Walker T., Johnson P.H., Moreira L.A., Iturbe-Ormaetxe I., Frentiu F.D., et al. (2011) “The wMel Wolbachia strain
blocks dengue and invades caged Aedes aegypti populations”, Nature 476: 450-453.
Walden P.M., Halili M., Archbold J.K., Lindahl F., Fairlie D., Inaba K., Martin J.L. (2013) “The alpha-proteobacteria
Wolbachia pipientis protein disulfide machinery has a regulatory mechanism absent in gamma-proteobacteria”, PloS
One, 8 11: e81440.1-e81440.9.
89
Disease resistance signaling in plant innate immune receptors, it’s
all about dimerisation
Simon Williams1, Kee Hoon Sohn2, Li Wan1, Maud Bernoux3, Panagiotis F. Sarris2, Cecile
Segonzac2, Thomas Ve1, Yan Ma2, Simon B. Saucet2, Daniel J. Ericsson1, Lachlan Casey1,
Xiaoxiao Zhang1, Anne Coerdt4, Jane Parker4, Peter Dodds3, Jonathan Jones2 and Bostjan
Kobe1
1
School of Chemistry and Molecular Biosciences, University of Queensland, QLD
Sainsbury Laboratory, John Innes Centre, Norwich, United Kingdom
3
CSIRO Plant Industry, Canberra, ACT
4
Max-Planck Institute, Cologne, Germany
2
A plant’s ability to detect and resist the infection of a specific pathogen rests with two critical
genes; a resistance (R) gene in the plant and a corresponding avirulence (effector) gene in the
pathogen. The protein products of R gene’s (immune receptors) play a surveillance role
within the plant cell and stimulate defence signaling after recognition of a specific effector
protein. The most predominant class of immune receptors encode tridomain proteins with a
central nucleotide-binding (NB) domain, a C-terminal leucine rich repeat (LRR) and either a
Toll-interleukin 1 receptor-like (TIR) domain or a coiled-coil (CC) domain at their Nterminus. The N-terminal TIR and CC domain are known to act as the signaling domain upon
activation of the R protein. Recently, homo-dimerisation of both TIR and CC domains was
reported as a crucial component during signaling activation (1, 2). We have used a
combination of protein crystallography and small-angled X-ray scattering to investigate this
further. Our structural investigations combined with functional data highlight the importance
of both homo- and heterodimeric interactions during disease resistance signaling. These data
help further our understanding of the steps required in the activation of plant innate immune
receptors.
References
[1]
[2]
Bernoux M., Ve T., Williams S., Warren C., Hatters D., Valkov E., Zhang X., Ellis J.G., Kobe B., Dodds P.N. (2011)
“Structural and functional analysis of a plant resistance protein TIR domain reveals interfaces for self-association,
signaling, and autoregulation”, Cell Host Microbe 9:200-211.
Maekawa T., Cheng W., Spiridon L.N., Toller A., Lukasik E., Saijo Y., Liu Shen Q-H., Micluta M.A., Somssich I.E.,
Takken F.L.W., Petrescu ., Chai J., Schulze-Lefert P: (2011) “Coiled-coil domain-dependent homodimerization of
intracellular barley immune receptors defines a minimal functional module for triggering cell death”, Cell Host
Microbe 9:187-199.
90
Crystal structure of a flax cytokinin oxidase and interaction studies
with a fungal effector
Li Wan1, Markus Koeck2, Simon Williams1, Peter Dodds2, Jeffrey Ellis2 and Bostjan Kobe1, 3
1
School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland,
4072, Australia
2
Plant industry, CSIRO, Canberra, ACT, 2601, Australia
3
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of
The flax rust effector AvrL567-A has been shown to physically interact with the flax
resistance (R) protein L6 [1]. This interaction activates the L6 protein, which results in the
initiation of the hypersensitive response defense pathway [2]. Using yeast-2-hybrid assays and
bimolecular fluorescence complementation assays in planta, we have identified another host
protein, the cytokinin oxidase LuCKX1.1, to interact with AvrL567-A. LuCKX1.1 is closely
related to AtCKX7, one of seven cytokinin oxidases in Arabidopsis thaliana responsible for
irreversible degradation of cytokinins [3]. Cytokinins are plant hormones involved in cell
division and a variety of developmental events, as well as plant immunity [4]. Currently, the
function of AvrL567-A during flax rust infection is unknown and we are interested in
determining if AvrL567-A targets LuCKX1.1 to promote pathogen virulence. Pathogen
infections have been shown to manipulate plant cytokinin levels so as to interfere with plant
immunity [5]. Interestingly, kinetic analysis revealed that the enzyme activity of LuCKX1.1
towards the cytokinins 2iP and trans-zeatin increases in the presence of AvrL567-A.
Compared with the wide-type, the AvrL567-A transgenic flax plant adopts a dwarfed
phenotype with a larger root system, which is typically associated with imbalanced cytokinin
levels in vivo. We determined the crystal structure of LuCKX1.1 at a resolution of 1.8 Å.
Utilizing the structure of LuCKX1.1 and the previously determined AvrL567-A structure, we
are investigating the interaction interface between the two proteins to help understand how
the interaction affects LuCKX1.1 enzyme activity. We anticipate that this combined
structural and biochemical study will provide insights as to the function of AvrL567-A during
infection and the role of LuCKX1.1 in disease resistance and/or susceptibility. The work will
be the first to report the molecular basis of how pathogen utilizes its effector protein to
manipulate plant cytokinin levels.
References:
[1] Dodds, P. N., Lawrence, G. J., Catanzariti, A. M., Teh, T., Wang, C. I., Ayliffe, M. A., Kobe, B. and Ellis, J. G. (2006)
[2]
[3]
[4]
[5]
“Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust
avirulence genes”, Proceedings of the National Academy of Sciences of the United States of America 103, 8888-8893.
Wang, C. I., Guncar, G., Forwood, J. K., Teh, T., Catanzariti, A. M., Lawrence, G. J., Loughlin, F. E., Mackay, J. P.,
Schirra, H. J., Anderson, P. A., Ellis, J. G., Dodds, P. N. and Kobe, B. (2007) “Crystal structures of flax rust avirulence
proteins AvrL567-A and -D reveal details of the structural basis for flax disease resistance specificity” Plant Cell 19,
2898-2912.
Bae, E., Bingman, C. A., Bitto, E., Aceti, D. J. and Phillips, G. N., Jr. (2008) “Crystal structure of Arabidopsis thaliana
cytokinin dehydrogenase”, Proteins 70, 303-306.
Hwang, I., Sheen, J. \and Muller, B. (2012) “Cytokinin signaling networks”, Annu Rev Plant Biol 63, 353-380.
Reusche, M., Klaskova, J., Thole, K., Truskina, J., Novak, O., Janz, D., Strnad, M., Spichal, L., Lipka, V. and
Teichmann, T. (2013) “Stabilization of cytokinin levels enhances Arabidopsis resistance against Verticillium
longisporum”, Molecular plant-microbe interactions: MPMI 26, 850-860.
91
Plants vs. pathogens: structural and functional studies of
Arabidopsis resistance protein SNC1 TIR domain and flax rust
effector protein AvrP
Xiaoxiao Zhang1, Simon Williams1, Thomas Ve1, Peter N. Dodds2, Jeffrey G. Ellis2 and
Bostjan Kobe1
1
School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre and
Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia
2
CSIRO Plant Industry, Canberra, Australian Capital Territory 2601, Australia
Plants are engaged in a contunuous battle against plant pathogens, which affects human
activities, especially our agriculture system. As an outcome of these battles, plants have
evolved a sophisticated immune system to detect pathogens. In this system, plant resistance
(R) proteins recognize pathogen proteins (effectors) in a highly specific manner, which leads
to the effector-triggered immune response (ETI) [1]. The biological functions of effector
proteins and the molecular bases of how R proteins are activated and signal are poorly
understood. A major sub-family of R proteins contain a Toll/interleukin-1 receptor (TIR)
domain at the N-terminus. Strucutral and functional analyses of the R proteins L6 and RPS4
have previously shown that the TIR domain region is both necessary and sufficient for
triggering ETI, and that TIR domain self-association is required for signalling [2]. Here we
report the crystal structure of the TIR domain from the Arabidopsis R-like protein SNC1.
Analysis of the structure combined with site-directed mutagenesis reveals two distinct
dimerization interfaces. Both interfaces have recently been shown to be important for
signalling, but this is the first time that these two interfaces have been shown to exist in the
same R protein and our structure provides a model for higher order TIR domain
oligomerization.
The molecular functions of most fungal effector proteins have not been identified. AvrP from
flax-rust is recognised by the flax reistsence protein P and is a small secreted cysteine-rich
protein of unknown function with sequence similarity to disulfide-containing Kazal protease
inhibitors [3]. However, homology modeling and biochemical studies suggest that AvrP is
not disulfide-bonded and may be strucutrally similar to plant homeodomain (PHD) zinc
finger proteins. Using zinc as an addtitive, we obtained crystals of AvrP diffracting to 2.5 Å
resolution and determined the three-dimentional structure using experimental phasing. The
structure of AvrP reveals a novel zinc-binding fold with some limited similarities to DNAbinding proteins. The zinc-coordinating region of the structure displays a positively charged
surface. The polymorphic residues in the AvrP family that are associated with R protein
recognition differences map to the surface of AvrP and lead to significant changes in surface
chemical properties in AvrP variants. The structure of AvrP provides insights into possible
pathogen-associated function of AvrP proteins, the mechanism of fungal infection in plants
and the plant-associated immune response.
The studiess in both plant resistance proteins and pathogen effetors bring us a step closer to
understanding the molecular basis for the resistance mechanisms in plants and the ability to
engineer novel resistance specificities for crops.
References
[1] Dodds, P.N. and Rathjen J.P. (2010) "Plant immunity: towards an integrated view of plant-pathogen interactions", Nat
Rev Genet, 11(8): 539-48.
[2] Bernoux, M., et al. (2011) "Structural and functional analysis of a plant resistance protein TIR domain reveals
interfaces for self-association, signaling, and autoregulation", Cell Host Microbe, 9(3): 200-11.
[3] Catanzariti, A.M., et al. (2006) "Haustorially expressed secreted proteins from flax rust are highly enriched for
avirulence elicitors", Plant Cell, 18(1): 243-56.
92

List of Abstracts – updated 4 April 2014

Transcription

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