COMPUTATIONAL ANALYSIS OF SURFACE PROPERTIES OF EF

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BIOPHYSICS
COMPUTATIONAL ANALYSIS OF SURFACE PROPERTIES
OF EF-HAND CALCIUM BINDING PROTEINS
DANA CRACIUN1, ADRIANA ISVORAN2
1
Teacher Training Department, West University of Timisoara, 4 V.Pirvan, 300223 Timisoara,
Romania, Email: [email protected]
2
Department of Biology-Chemistry, West University of Timisoara, 16 Pestalozzi, 300316 Timisoara,
Romania, Email: [email protected]
Received August 14, 2013
Within present study we perform a computational analysis of the surface properties of
the EF-hand calcium binding proteins (EFCaBPs), both at global and local levels.
Among EFCaBPs there are calcium sensors involved in signal transduction processes
and exhibiting extended spatial structures and calcium buffering proteins exhibiting
compact structures. Structures superposition reflects higher structural similarity
between extended forms, the compact ones being more divergent in good correlation
with their sequence alignment. Surfaces of extended EFCaBPs present a smaller
number of cavities but with larger volumes and areas than compact ones in correlation
with their known biological functions. Surface electrostatic potential is higher for
extended EFCaBPs, underlying the role of electrostatics repulsions in adopting their
spatial structures and also the possible role in binding charged peptides.
Key words: calcium binding proteins, surface, electrostatic potential.
1. INTRODUCTION
Calcium ions are indispensable for the physiology of living cell being
involved in many cellular processes. The key role of calcium ions strongly depends
on a large number of proteins able to bind them, so-called calcium binding
proteins, CaBPs [1]. The group of CaBPs is wide and heterogeneous. There are
membrane intrinsic CaBPs acting as calcium transporters and involved in the
control of calcium ions concentration, calcium buffers and calcium-modulated
proteins involved in signal-transduction processes [2].
Calcium modulated proteins may be found both in extra- and intracellular
environment and from structural point of view they may be divided in two families
[3]: CaBPs containing EF-hand motifs and CaBPs lacking EF-hand motifs
respectively.
Rom. Journ. Phys., Vol. 59, Nos. 3–4, P. 339–345, Bucharest, 2014
340
Dana Craciun, Adriana Isvoran
2
Within this study we fix our attention on the EF-hand calcium binding
proteins. The EF-hand motif is a structural domain containing a calcium binding
loop twelve residues long flanked on both sides by an alpha-helix also twelve
residues long [3].The biggest part of CaBPs enclose two domains, N- and Cterminal, each containing two EF-hand motifs and connected by a linker region [4].
Among EF-hand CaBPs there are calcium sensors involved in signal
transduction processes and exhibiting open, extended spatial structures and calcium
buffering and calcium transporting proteins exhibiting closed, compact structures
[5]. The main structural difference between the two categories of EF-hand CaPBs
[4] is displayed by the terminal domains linker region that is structured in a straight
helix for the calcium sensors (as it is revealed in figure 1 for rabbit skeletal muscle
troponin, black) and is unstructured for calcium buffers and calcium transporting
proteins (figure 1 for bovine neurocalcin delta - grey). These structures have been
retrieved from the Protein Data Bank [6]. Structural dissimilarity of the two
categories of EF-hand CaBPs is reflected in their distinct biological functions.
Fig. 1 – Superpostion of two structures of EFCaBPs: the extended form of rabbit skeletal muscle
troponin in black (PDB code entry 1TN4) and the compact form of bovine neurocalcine delta in grey
(PDB entry code 1REC).
Tacking into account the involvement of CaBPs in many diseases and the fact
that not all of them have determined spatial structures, it becomes important to
predict their spatial organizations and their structural and functional properties,
respectively.
There are in specific literature a few studies concerning the physicochemical
factors determining the extended or compact structure of the EF-hand CaBPs [7–9]
revealing the involvement of electrostatics interactions [7–8] and the essential role
3
Computational analysis of surface properties of EF-hand calcium binding proteins
341
of the hydrophilicity of resides linking the domains [9]. Also, the concepts of the
fractal geometry have been applied to investigate their global structural properties
[10–11] illustrating distinct fractal properties of backbones of extended respective
compact structures [10] and distinct scaling properties of radius of gyration and
surface area of the two categories of EF-hand CaBPs [11] confirming the dissimilar
mechanisms responsible for their global folding.
Within present study we perform a computational analysis of the surface
properties of the two categories of EF-hand CaBPs both at global and local levels
and interpret them in terms of specific biological interactions.
2. METHODS
Experimentally determined structures of proteins are archived in the Protein
Data Bank [6] that we have used in order to retrieve the structural files of
EFCaBPs. There are numerous structures of EFCaBPs deposited in PDB, but, with
the exception of human centrin 2 (that has experimentally-determined structure
only in the presence of a peptide) we have considered in our study only those
structures obtained using X-ray diffraction technique and containing the entire
protein and the bound calcium ions where it was the case. These proteins are
presented in the Table 1.
Table 1
The investigated proteins
Protein
PDB code
Conformation
1CLL
4TNC
Number of amino acids
in structural file
143
159
Human calmodulin
Chicken skeletal muscle
troponin C
Rabbit skeletan muscle troponin
C
Paramecium tetraurelia
calmodulin
Potato calmodulin
Human calmodulin-like protein
Human centrin 2
Bovine recombinant neurocalcin
delta
Rattus norvegicus calcineurin B
Bos Taurus recoverin
Amphioxus sarcoplasmic
calcium-binding protein
Nereis diversicolor sarcoplasmic
calcium-binding protein
1TN4
157
Extended
1OSA
148
Extended
1RFJ
1GGZ
2GGM
1BJF
148
140
144
181
Extended
Extended
Extended
Compact
2CT9
1REC
2SAS
195
191
185
Compact
Compact
Compact
2SCP
174
Compact
Extended
Extended
342
4
Multiple sequence alignment of the considered proteins has been done using
CLUSTALW [12], a free accessible on-line tool.
In order to analyze the surface properties of considered proteins we have used
a few computational tools: CHIMERA [13] for surface area calculation and
structures superposition, PyMol [14] for electrostatic potential computation and
CASTp [15] for cavities identification and characterization. The distribution of
physico-chemical properties along the protein chain is obtained using free on-line
available tool ProtScale [16].
3. RESULTS AND DISCUSSIONS
Sequence alignment of considered proteins reveals high sequence identity
between extended EFCaBPs (more than 73%) and a low sequence identity between
extended and compact ones (lower than 47%, data not shown), the calcium binding
loops sequences being the most conserved in both extended and compact
structures. This remark is also confirmed by structures superposition for the
considered EFCaBPs. The superposition of extended structures reveals a high
structural similarity between them, the structural motif that is almost identical in all
these proteins being the domains linker region. For the compact EFCaBPs, the
structure superposition indicates a small structural identity. Also, there is low
structural identity between extended and compact EFCaBPs.
The structural files of considered proteins and their relevant surface
properties are presented in Table 2.
Table 2
The surface properties of the considered proteins
PDB
code
Number
of cavities
1CLL
4TNC
1TN4
1OSA
1RFJ
1GGZ
2GGM
1BJF
2CT9
1REC
2SAS
2SCP
16
17
15
17
14
20
18
24
29
23
19
36
Volume of the
biggest cavity
[Å3]
1941,4
581,3
489
2135,1
255,7
274,7
2258,8
484,6
466,6
306,1
1274,9
259,6
Surface of biggest
cavity
[Å2]
581,8
241,6
296,5
621,2
180,9
148,4
1286,5
323,7
329,1
168,4
801,4
271,7
Surface electrostatic potential
[kbT/e] (T=300˚K)
-102.38÷102.38
-102.38÷102.38
-100.44÷100.44
-97.18÷97.18
-97.89÷97.89
-94.72÷94.72
-106.19÷106.19
-67.99÷67.99
-80.37÷80.37
-80.48÷80.48
-78.74÷78.74
-76.09÷76.09
5
343
Surfaces of extended EFCaBPs present a smaller number of cavities but with
larger volumes and areas than compact ones, in good correlation with their known
biological functions [1, 3]. Surface electrostatic potential is higher for extended
EFCaBPs, underlying the role of electrostatics repulsions in adopting their spatial
structures [7] and also their possible role in binding charged peptides.
As these proteins contain distinct numbers of amino acids, in order to
compare their surface areas we have considered the surface area per amino acid by
dividing the total surface area to the amino acid number. Figure 2 shows that
surface area per amino acid for extended forms (medium pattern) and compact
forms (dense pattern) are distinct.
Fig. 2 – Surface area per amino acid for extended forms (medium pattern)
and compact forms (dense pattern).
There is a statistically difference between the two means, (46.39±7.2 ) Å2 for
extended forms and (54.46±6.5) Å2 for compact ones, reflected by t- and one-way
ANOVA tests and underlying the distinct degree of density of their tertiary
structures.
It is already known that extended EFCABPs present a large solvent exposed
hydrophobic surface that interacts with targets [17] and these surface is mainly
build by the linker composition in amino acids. Figures 3 present the average
hydrophobicity respective the accessible surface area for the linker region of rabbit
troponin C, an extended EFCaBP (A) and the same distributions for bovine
recombinant neurocalcin delta (B), a compact EFCaBP. The diagrams are obtained
using ProtScale tool [16].
344
6
1TN4
3
180
average hydrophobicity
accesible surface area
160
140
120
1
100
80
0
60
40
-1
20
2
0
-20
-2
74 76 78 80 82 84 86 88 90 92 94 96 98 100 102 104 106
amino acid position
(A)
1BJF
3
160
140
120
100
1
80
60
0
40
20
-1
0
accesible surface areea
2
-20
-2
84
86
88
90
92
94
96
98 100 102 104 106 108 110
amino acid position
(B)
Fig. 3 – The distribution of average hydrophobicity respective the accessible surface area
for the linker region of rabbit troponin C (A) and of bovine recombinant neurocalcin delta (B)
The profiles of average hydrophobicity and surface accessible area are
distinct and opposite for the rabbit troponin C and they are quite similar in the case
of bovine neurocalcin delta. It reflects that the two types of CaBPs expose to the
solvent distinct hydrophobic surfaces, in correlation with their distinctive
biological functions.
7
345
4. CONCLUSIONS
The interaction between any two proteins is strongly dependent on the
properties of their interafaces. Present studies emphasize distinct global and local
surface properties of the two families of EFCaBPs concerning the distribution of
surface electrostatic potential, the presence of cavities and their distinct properties
(dimensions and hydrophobicity). It suggests that there are distinct and specific
ligands to be used to inhibit their pathological interactions.
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COMPUTATIONAL ANALYSIS OF SURFACE PROPERTIES OF EF

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