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BIO DESIGN
MINI REVIEW P 1-13
Two component signaling systems in
Mycobacterium tuberculosis
Ha Yeon Cho and Beom Sik Kang*
School of Life Sciences and Biotechnology, Kyungpook National University, Daegu 702-701, Korea.
*Correspondence: [email protected]
Mycobacterium tuberculosis is one of fearful pathogens and has the ability to persist within its host. Its successful survival
is due to alterations in gene expression in response to environmental changes by two component systems (TCS), which
consist of sensor histidine kinases (HK) and their cognate response regulators (RR). M. tuberculosis has twelve TCSs and
five orphan RRs. A typical TCS involves sensing of internal or external signals by a HK, leading to its autophosphorylation,
followed by phosphoryl transfer to the cognate RR, which functions as a transcriptional activator. To understand the
function and the mechanism of M. tuberculosis TCSs, the components of HKs and RRs are subjected on the structural
studies and the results could be useful for new antituberculosis drug development. Here, structural features of HKs and
RRs currently revealed from M. tuberculosis are summarized. Those include GAF and PAS domains for senor domains and
ATP binding domains from HKs, and the receiver and effector domains from RRs.
INTRODUCTION
The tubercle bacillus Mycobacterium tuberculosis was first
identified and described in 1882 by Robert Koch. Tuberculosis
(TB) is a chronic infectious disease caused by M. tuberculosis,
and is spread through the air from one person to another by
coughing and sneezing. Currently one-third of the world’s
population is thought to have been infected with this bacillus
with new infections occurring in about 1% the population each
year (Parrish et al., 1998). Those people carry the bacterium
in the dormant state and the pathogen is insensitive to most
available chemotherapy in this state. It is why the tuberculosis is
more difficult to complete cure. Moreover, due to improper use
of drugs in chemotherapy, the drug resistance arises. Recently,
TB drug resistance is an important public health problem that
threatens progress made in TB care and control worldwide.
In addition to multidrug-resistant TB, there is emergence of
extensively drug-resistant TB, which is resistant to most of
drugs including isoniazid, rifampin, any fluoroquinolone and the
second-line drugs (Falzon et al., 2011). Moreover, the synergy
created between TB and AIDS makes each disease considerably
more deadly (Hopewell, 1992). M. tuberculosis can persist within
the human host for years without causing disease, in a syndrome
known as latent TB. The mechanisms by which M. tuberculosis
establishes a latent metabolic state, eludes immune surveillance
and responds to triggers that stimulate reactivation are a high
priority for the future control of TB (Parrish et al., 1998).
TB infections are normally localized in the lungs, which indicate
that normal in vivo growth and survival requires oxygen. However,
mycobacteria also experience hypoxic conditions in vivo inside
macrophages. Thus, M. tuberculosis growth is inhibited when
the bacilli are engulfed into granulomas, the inside of which
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is associated with low oxygen tension. M. tuberculosis enters
dormancy in response to hypoxia or exposure to nitric oxide from
macrophage. Sensing and responding to environmental stimulus
is a critical for cell survival and growth. In bacteria, adaptation
to environmental signals is mediated primarily through serinethreonine protein kinases, extra-cytoplasmic function sigma
factors, and two-component signal transduction systems
(Ashby, 2004). Two-component system (TCS) has been shown
to regulate many physiological processes, including sporulation,
competence, antibiotic resistance, transition into stationary
phase, and virulence (Krell et al., 2010). The TCS is composed
of a homodimeric sensor histidine kinase (HK) and its cognate
response regulator (RR) protein (Laub and Goulian, 2007). In
the prototypical TCS, a HK consists of sensor core, which
receives environmental stimuli and kinase core, which transfers
the signal to the cognate RR by means of phosphorylation.
Kinase core binds ATP through its ATP binding domain (ABD)
and autophosphorylates a conserved His in its dimerization
and histidine phosphotransferase (DHp) domain (Dutta et al.,
1999). The phosphoryl group is then transferred to an Asp in
the receiver domain of the RR, activating its output domain to
effect cellular changes, often through changes in transcription
(Figure 1). Basic two component phosphoryl transfer signal
transduction pathway involves three phosphotransfer reactions
(autophosphorylation, phosphotransfer, and dephosphorylation)
and two phosphoprotein intermediates (phosphorylated HK and
phosphorylated RR)(Stock et al., 2000).
TCSs are ubiquitous in bacteria while it only presents in some
plants, lower eukaryotes, and archaea (Kim and Forst, 2001). The
importance of TCSs in bacterial survival and the absence of TCSs
in higher eukaryotes also make these systems attractive targets
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Two component signaling systems in Mycobacterium tuberculosis
FIGURE 1 I Two-component signal transduction system. The prototypical two-component system consists of a histidine kinase (HK) and a response
regulator (RR). The HK contains a sensor domain recognizing an external signal, DHp domain, at which a conserved H residue is, and ATP binding domain
(ABD) while RR consists of receiver domain (REC) and effector domain. Autophosphorylation occurs in the HK, followed by phosphoryl group transfer to a
conserved D residue of the RR. A phosphorylated RR binds to a target DNA to regulate downstream genes (Dutta et al., 1999; Francis et al., 2013).
for therapeutic development against pathogenic organisms
(Barrett and Hoch, 1998). Consistent with this idea, mutant
strains that are defective in specific TCSs that lead to virulence
attenuation are now being investigated as potential vaccine
candidates. M. tuberculosis has evolved several mechanisms
to circumvent the hostile environment of the macrophage, its
primary host cell (Wayne and Hayes, 1996). Understanding
the molecular mechanisms of M. tuberculosis pathogenesis
using TCS will provide insights into the development of targetspecific drugs or effective vaccine candidates for the treatment
of the disease (Meena and Rajni, 2010). In this review, we
summarize current studies on the protein members of TCSs in M.
tuberculosis in the structural aspect.
TWO-COMPONENT SYSTEMS IN
M. TUBERCULOSIS
A TCS, MtrA-MtrB system is the first such system to be
characterized in the tubercle bacillus (Curcic et al., 1994).
Since then, sensor HKs and RRs have been identified in the M.
tuberculosis H37Rv genome. Genomic analysis indicates that M.
tuberculosis encodes twelve complete TCSs and five remaining
potential orphan RR or HK proteins. The TCS are summarized in
Table I with their genomic location tags and their nomenclatures.
Those are named based on close homologs of known or
postulated function (Cole et al., 1998; Morth et al., 2004). M.
tuberculosis contains few two-component systems compared
to many other bacteria such as Bacillus subtilis and Escherichia
coli, in which there are more than thirty different two-component
regulatory systems (Kunst et al., 1997). The number of intact
TCSs in M. tuberculosis is lower than that typically found in other
bacteria of similar genome size, possibly reflecting the evolution
of this bacterium as a strict human pathogen and its adaptation
to a predominantly intracellular lifestyle (Cole et al., 1998).
MtrA-MtrB system identified in 1994 (Curcic et al., 1994). The
RR MtrA modulates M. tuberculosis proliferation in macrophages.
This TCS regulates essential physiological processes, including
2
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DNA replication and cell wall integrity (Zahrt et al., 2000). The
SenX3-RegX3 system was identified as an inorganic phosphatedependent regulator of genes involved in phosphate acquisition
(Glover et al., 2007). It was known to responds to phosphate
starvation. The PhoP-PhoR system responds to intracellular pH
and regulates biosynthesis of complex lipids. It is implicated in
regulating production of complex cell wall lipids (Abramovitch et
al., 2011). The PrrA-PrrB TCS plays a role in early adaptation to
intracellular infection (Ewann et al., 2002) and is essential for in
vitro survival (Ewann et al., 2004). Transcription of the genes prrA
and prrB is induced under nitrogen limitation and repressed in
hypoxia (Haydel et al., 2012). MprA-MprB module responds to
stress and regulates expression of the alternative sigma factors
(SigB and SigE)(He et al., 2006).
Very little is known about the biochemical activity or functional
significance of the NarL-NarS TCS. NarL is homologous to those
of NarL from E. coli (Schnell et al., 2004), the RR component of
the NarQ-NarL TCS. This system regulates genes in response
to nitrate concentrations (Stewart, 1993). KdpD-KdpE as
homologs in other bacteria, KdpD interacts with the regulator
KdpE. KdpDE controls the expression of the adjacent kdpFABC
operon in response to K + concentration (Steyn et al., 2003).
TrcR-TrcS system is expressed during aerobic growth in culture
and at low levels early after infection of human macrophages
(Haydel et al., 2002). Two ORFs encoding RR (TcrX) and HK
(TcrY) are conserved in all species of Mycobacteria except
Mycobacterium leprae (Tyagi and Sharma, 2004). The genes tcrX
and tcrX are significantly induced under iron-limitation (Bacon
et al., 2003) and during the post-infection period (Haydel et al.,
2004), respectively. The structure and sequence analysis suggest
that the PdtaS-PdtaR system is structurally equivalent to the
EutW-EutV system regulating ethanolamine catabolism in some
organisms (Preu et al., 2012).
Among the HKs, DosS and DosT function with a single RR,
DosR, which functions to up-regulate genes essential for the
survival of M. tuberculosis under hypoxic conditions. About 48
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Ha Yeon Cho and Beom Sik Kang
TABLE 1 I Two-component systems in Mycobacterium tuberculosis
HK/RR
ORF annotation
Signal
Function
Reference
SenX3/RegX3
Rv0490/Rv0491
O2, NO, CO Phosphate
(Singh et al., 2014)
(Himpens et al., 2000)
U/U/TcrA
Rv0600c/Rv0601c/
Rv0602c
Tetrahydrolipstatin
Oxygen sensing
Virulence, phosphate uptake, aerobic
respiration
Involved in small molecule metabolism
PhoR/PhoP
Rv0758/Rv0757
pH
Cell wall components
(Abramovitch et al., 2011)
NarS/NarL
Rv0845/Rv0844c
Unknown
Unknown
(Parish et al., 2003)
PrrB/PrrA
Rv0902c/Rv0903c
Macrophage infection
Adaptation to intracellular infection
(Ewann et al., 2004)
MprB/MprA
Rv0982/Rv0981
Detergents
Response to stress conditions
(Zahrt et al., 2001)
KdpD/KdpE
Rv1028c/Rv1027c
Possibly [K]
Predicted potassium uptake system
(Parish et al., 2003)
TrcS/TrcR
Rv1032c/Rv1033c
Unknown
Unknown
(Haydel et al., 1999)
DosS/DosR
(DosT/DosR)
Rv3132c/Rv3133c
(Rv2027c/Rv3133c)
Low O2, NO, CO
Hypoxia sensing
(Saini et al., 2004a and b)
MtrB/MtrA
Rv3245c/Rv3246c
Unknown
DNA replication and cell wall integrity
(Zahrt et al., 2000)
TcrY/TcrX
Rv3764c/Rv3765c
Low iron, starvation
Involvement in virulence
(Parish et al., 2003)
PdtaS/PdtaR
Rv3220c/Rv1626
Unknown
Unknown
(Morth et al., 2005)
genes of M. tuberculosis were reported to be induced under
hypoxic conditions, as well as on exposure to nitric oxide (Park
et al., 2003). The up-regulation of these genes is regulated by the
DosS-DosR system (Wayne and Hayes, 1996). Although DosT is
structurally very similar to DosS, DosT works as a direct oxygen
sensor while DosS functions as a redox sensor (Kumar et al.,
2007). Unlike other two component system, a putative HK in M.
tuberculosis separated into two proteins, HK1 (Rv0600c) and
HK2 (Rv0601c), makes a unique three-protein system with a RR,
TcrA (Rv0602c). HK1 and HK2 are both annotated as putative
HKs that phosphorylate TcrA (Shrivastava et al., 2006). HK2
gene is up-regulated in response to tetrahydrolipstatin, an antimicrobial agent (Waddell et al., 2004).
There are five orphan RRs found in the genome of M.
tuberculosis. The genes for those proteins are Rv0195, Rv0260c,
Rv0818, Rv2884, and Rv3143. Rv0195 is similar to the member
of the LuxR family, and seems to be induced by sodium azide
treatment (Boshoff et al., 2004) or nutrient starvation (Betts et al.,
2002). Rv0260c has a transcriptional regulatory motif of RRs but
also has homology with HemD, an uroporphyrinogen-II synthase.
Rv0818 is a homolog of GlnR involved in regulation of nitrogen
metabolism, while Rv2884 is an RR of unknown function. In
addition, Rv3143 has a receiver domain, but no effector domain,
suggesting it may play a role in a phosphorelay system (Zhou et
al., 2012).
HISTIDINE KINASES
HKs are usually membrane proteins, composed of the N-terminal
sensor domain, which receives the specific signal, and the
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(Waddell et al., 2004)
C-terminal kinase domain as a transmitter domain (Dutta
et al., 1999). The specificity of the system lies in the sensor
domain, which recognizes changes in the environment. As
HKs can respond to various stimuli, a wide range of sensor
domains are found in the periplasmic portions of membranebound HKs (Cheung and Hendrickson, 2010). However, the
kinase domain is largely conserved and provides both the site
of autophosphorylation and the interaction domain with the RR
in cytosol. The C-terminal kinase domain of HKs is generally
composed of a DHp domain and a CA domain (Dutta et al.,
1999). The DHp domain is the interaction site between two
monomers of the HK and is the interface to the RR. Thus, it
controls the specificity of the HK and RR interaction (Marina
et al., 2005). In HKs, the DHp and CA domains are shown
with common intracellular domains PAS (Per Arnt Sim), HAMP
(histidine kinase, adenyl cyclase, methyl-accepting proteins, and
phosphatase), and GAF (cGMP-specific phosphodiesterase,
adenylyl cyclase, and FhlA). Some HKs have multiple copies of
such domains (Figure 2a~g).
SENSOR DOMAINS
Sensor domains of HKs are located outside of cell membrane,
in membrane or in cytosol. Currently, there is little structural
information of the sensor domains embedded in membrane
(Mascher et al., 2006). The prototypical HK has an extra-cytosolic
sensor domain that senses extracellular signals or conditions
in the cell envelope. These sensor domains have highly diverse
sequences. However, most of the known structures of extracytosolic sensor domains fall into three distinct structural folds,
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Two component signaling systems in Mycobacterium tuberculosis
domain has α-β(2)-α(2)-β(3) structure
while GAF domain consists of α(2)-β(3)-αβ(3)-α. The GAF-A domain has a relatively
long loop between strands β3 and β4
(a)
(b)
compared with the PAS domain while the
canonical GAF domain has an α-helix
in the corresponding loop. Although,
topologically, the GAF-A domain of DosS
is similar to a PAS domain, its overall
folding is that of the canonical GAF
domain. However, the canonical GAF
domain has a curved six-stranded β-sheet
(c)
(d)
(e)
(f)
(g)
(h)
FIGURE 2 I Domain organizations of histidine kinases and a response regulator in Mycobacterium
tuberculosis. All histidine kinases (a ~ g) has conserved domains for dimerization and histidine
phosphotransferase (DHp) and ATP binding domain (ABD) at their C-terminus. Organization of the
N-terminal domains is variable. Typically, transmembrane proteins consisting of extra-cytoplasmic sensor
domains, transmembrane helices (TM), and HAMP (histidine kinase, adenyl cyclase, methyl-accepting
proteins, and phosphatase) domain. (h) Response regulator is composed of two domains, a conserved
N-terminal receiver (REC) domain and a variable C-terminal effector domain.
mixed α/β, all-helical, and β-sandwich (Cheung and Hendrickson,
2010). Unlike extra-cytosolic sensor domains, many cytosolic
sensor domains can be annotated on the sequence level as
PAS or GAF domains (Hefti et al., 2004; Martinez et al., 2002).
Sensor domains currently revealed in HKs of M. tuberculosis
are also GAF and PAS domains. The amino acid sequence and
spectroscopic analyses of SenX3 suggest it has a PAS domain
containing a heme as the sensor domain (Rickman et al., 2004).
DosS and DosT have two GAF domains in their sensor core. By
amino acid sequence analysis, they had been suggested to have
two transmembrane helices in their N-terminal domains implying
membrane anchoring. However, the crystal structures of GAF
domain revealed those helices are a part of GAF domains (Cho
et al., 2007; Podust et al., 2008). The N-terminal GAF and PAS
tandem domain structure of PdtaS is determined and expected
to play a role for sensor domain (Preu et al., 2012).
GAF domains play important roles as regulatory elements found
in many proteins from various organisms and are known to be
small molecule binding domains. Many GAF-containing proteins
have two GAF domains in tandem and the two domains have
separate functions, binding a cyclic nucleotide and dimerization
(Martinez et al., 2002 and 2005). The two GAF domains in the
M. tuberculosis DosS and DosT proteins are also arranged in
tandem and the first GAF domain (GAF-A) contains a heme
(Sardiwal et al., 2005). The GAF-A consists of the sheet and two
a-helices on each side of the sheet with the secondary structure
order of α-β(2)-α(2)-β(3)-α (Figure 3a). It is topologically similar to
the PAS domain found in the E. coli protein Dos (Figure 3b). Both
PAS and GAF domains belong to the Profilin-like domain family,
which have an α+β protein with α-β-α layers. The canonical PAS
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forming a half-barrel structure containing
a cyclic nucleotide, while the sheet of
GAF-A is five-stranded and rather flat (Cho
et al., 2007).
In the heme-bound GAF-A, a long
peptide region containing two helices
(α2 and α3) connects strands β2 to β3
at each end of the sheet. This peptide
crosses over the sheet with a space
existing between a loop connecting the
α2 and α3 helices and the sheet. The
space is covered by two loops connecting strands β1 to β2, and
strands β3 to β4 at the top and bottom positions completing the
inside cavity. A b-type heme is tightly packed into the cavity and
at the proximal position of the heme a histidine is provided from
a loop connecting the β3 and β4 strands. The plane of the heme
is roughly perpendicular to the sheet. It differs from that in the
PAS domain, in which a heme is inserted into a crevice formed
between an α-helix and the β-sheet. The plane of the heme is
parallel to the β-sheet (Figure 3a, 3b) (Cho et al., 2007).
DosT is structurally similar to DosS and also has a heme at
its GAF-A. The heme group is embedded in a defined space
surrounded by hydrophobic residues and the route from outside
of the protein to the iron in heme is limited to a channel in DosS
and DosT GAF-As. The structure of DosT GAF-A reveals a wide
pore on the protein surface providing a potential route for the
access of O2 to the sensing pocket (Podust et al., 2008). DosT
GAF-A has a wide-open channel while DosS GAF-A has a
narrow, bent channel. In DosS GAF-A, the channel to the heme
iron is completely blocked by the side chain of Glu87 in the
middle of the channel when it faces the heme. The channel in
DosT GAF-A is always open due to the absence of a side chain
at Gly85 (corresponding to Glu87 in DosS GAF-A) (Figure 3d,
3e). The elimination of the side chain in DosS GAF-A to open
the channel to the heme by mutations of Glu87 to Ala or Gly
increases the accessibility of O 2 to the heme. Although DosS
is structurally similar to DosT, which is a direct oxygen sensor,
DosS GAF plays as a redox sensor because it loses its electron
before O2 approaches the heme (Cho et al., 2011). In hypoxic
condition, DosT loses O2 through the widely open channel, while
DosS senses this condition through the reduction of heme to
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Ha Yeon Cho and Beom Sik Kang
(a)
(d)
(b)
(e)
(c)
(f)
FIGURE 3 I Structures of cytosolic sensor domains. (a) Ribbon diagram of DosS GAF-A showing an α-β-α layer with five-stranded antiparallel β-sheet. The
plane of the heme is perpendicular to the sheet (Cho et al., 2009). (b) A PAS domain containing a heme from Escherichia coli. The plane of the heme is parallel to
the β-sheet (Cho et al., 2009). (c) The GAF-B domain of DevS consists of a six-stranded antiparallel β-sheet and three α-helices. The structures from DosT (d) and
DosS (e) GAF-A are presented with ribbon and mesh, which are sectioned to show the internal channel. Heme and side chain of E87 are shown as sticks. Arrows
indicate the channel (Cho et al., 2011). (f) Ribbon diagram of the N-terminal sensor domain of PdtaS consisted of GAF and PAS domains. For the connectivity, the
invisible linker between GAF and PAS domain and the disordered region in the GAF domain are shown with purple and grey lines, respectively (Preu et al., 2012).
ferrous state by abundant reduced FAD due to the depletion of
final electron acceptor O2.
DosS from M. tuberculosis has been also called as DevS.
The structure of GAF-B domain of M. smegmatis DevS was
determined and the overall folding of similar to the GAF-A
domain of DosS. However, it has an additional strand in front
of the α3-helix completing the six-stranded β-sheet, which is a
key feature of a GAF-domain. The strongly curved β–sheet of
GAF-B forms a half barrel (Lee et al., 2008). This GAF domain
is structurally similar to the GAF domains from other proteins
containing cGMP, cAMP or biliverdin molecules in their binding
pockets (Wagner et al., 2005; Yang et al., 2007). Unlike these
GAF domains, a short loop of DevS GAF-B connecting strands
β4 and β5 does not contain the α-helix forming the binding
cavity in the other GAF domains. Two loops connecting the β2
and β3 strands and the β4 and β5 strands are located close
to the inside of the half-barrel structure. There is no space for
cyclic nucleotide binding in the structure of DevS GAF-B (Figure
3c). Unlike other GAF domains binding cAMP or cGMP, in which
several hydrophilic residues are involved in the binding, GAF-B
of DosS and DosT have hydrophobic residues in the positions
for the hydrophilic residues, implying that these GAF-B are not
suitable to bind a small ligand such as cyclic nucleotides (Lee et
al., 2008).
PdtaS is a cytosolic histidine kinase like DosS and DosT. It has
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GAF and PAS domains in tandem at its N-terminal sensor core
(Figure 3f). The structure of complete N-terminal sensor region
of PdtaS from M. tuberculosis reveals closely linked GAF and
PAS domains (Preu et al., 2012). PAS domains bind a variety of
substrates including chromophores, heme, and flavin nucleotides
(Taylor and Zhulin, 1999). In the structure of PdtaS PAS domain,
the internal cavity is apparently too small to hold a heme or other
small chromophores. The full-length PdtaS exists in equilibrium
between a monomeric and dimeric, and the N-terminal region of
the protein is known to form dimers. In the crystal a moleculemolecule interface suggests that the dimer interface consists
of PAS–PAS, GAF–GAF and PAS–GAF interactions. A dimeric
structure is in a head-to-tail arrangement; relative to the long axis
of the dimer, equivalent domains are at opposite ends of the long
axis. However, the C-termini of the domains are close together
allowing the formation of the coiled-coil structure of the DHp
domains (Preu et al., 2012).
By sequence analysis SenX3 is revealed to have a PAS domain
that in other proteins is known to function as an input module
that senses oxygen and redox potential (Rickman et al., 2005).
This PAS domain contains a heme and is presumed to work as a
sensor for an oxygen-controlled replication switch (Singh et al.,
2014). Null mutations in the sensor genes are indeed attenuated
but show a persistence phenotype (Rickman et al., 2004). The
virulence factor SenX3 is a heme protein that acts as a three-way
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Two component signaling systems in Mycobacterium tuberculosis
(a)
(b)
(c)
(d)
(e)
FIGURE 4 I Sequence alignment and structures of the ATP binding domains. (a) Amino acid sequences of thirteen ABDs from Mycobacterium tuberculosis
HKs and ABD from Bacillus subtilis DesK were compared. These are divided into two groups according to the presence or absence of an F box. PrrB, TrcS, TcrY,
PhoR, SenX3, MprB, MtrB, KdpD, and Rv0600c have an ATP lid (between the F and G2 boxes) and conserved F and R residues in their F boxes. DosS, DosT,
NarS, PdtaS, and DesK have short sequences between the G1 and G2 boxes. Secondary structural (blue helices and yellow strands) above the sequences are
based on DosS and PrrB structures for each group. Conserved amino acid residues are shaded grey, and conserved residues among the group members are
shown by red (Cho et al., 2013). PhoQ (PDB ID 1IDO) (b) and PrrB (PDB ID 1YSR) (c) show a typical two-layer α/β sandwich structure. The helices and the strands
are colored by red and yellow, respectively. The nucleotide is shown as sticks and the ATP lid is purple. Superimposition of the DosS ABD (green) (d) and DosT
ABD (blue) (e) on the PhoQ ABD (orange) bound to an ATP analog. The ATP is surrounded by the F box helix and long ATP lid. A loop between β3 and α3 in the
DosS and DosT ABD overlies the ATP binding site (Cho et al., 2013).
sensor with three levels of activity. The oxidation of SenX3 heme
by oxygen leads to the activation of its kinase activity, whereas
the deoxy-ferrous state confers a moderate kinase activity. The
binding of nitric oxide and carbon monoxide inhibits kinase
activity (Singh et al., 2014). Consistent with these biochemical
properties, the SenX3 mutant of M. tuberculosis is capable of
attaining a non-replicating persistent state in response to hypoxic
stress, but its regrowth upon the restoration of ambient oxygen
levels is significantly attenuated compared to the wild type and
the complemented mutant strains.
ATP BINDING DOMAINS
During the autophosphorylation reaction of the kinase core, the
ABD transfers a phosphate group from ATP to the His in the DHp
domain. The ABD not only binds ATP but also interacts with the
DHp dimer. The structures of the ABDs are well conserved in HKs
and it has α/β sandwich fold consisted of two layers, a layer of
mixed five-stranded β-sheet and a layer of three α-helices (Figure
4b). ABDs of HKs generally have conserved boxes for ATP
binding. Those are N box, F box, and three G boxes containing
the conserved residues, Asn, Phe, and Gly, respectively (Dutta
et al., 1999). ABDs also usually have an ATP lid, which covers
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the nucleotide. It is a long characteristic loop between the F and
G2 boxes and exhibits a variety of conformations in the absence
of nucleotide (Nowak et al., 2006; Marina et al., 2001). Charged
residues in the ATP lid are conserved for the interaction to the
nucleotide beta-phosphate. The ATP lid is known to play a role
for kinase activity and to protect ATP from futile reactions (Casino
et al., 2009).
The amino acid sequences alignment of ABDs with conceptually
translated ABDs of other known HK genes from M. tuberculosis
H37Rv, revealed that the domains can be divided into two
groups. One contains both the conserved F box and the ATP lid,
known to be involved in ATP binding, and the other is without
either of these motifs. The former group includes ABDs of PrrB,
TrcS, TcrY, PhoR, SenX3, MprB, MtrB, KdpD, and Rv0600c, and
those belong to the OmpR family. DosS, DosT, NarS, and PdtaS
belong to the latter group (Figure 4a). The ABD structures of PrrB
(Nowak et al., 2006), DosS and DosT ABD have been determined
among the HKs of M. tuberculosis (Cho et al., 2013).
Those structures have the canonical structure. PrrB ABD (Nowak
et al., 2006) consists of a single domain with a two-layer α/β
sandwich. A mixed five-stranded β sheet with the strand order of
2-4-5-7-6 is on one side and three α helices (α1, α2, and α3) are
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Ha Yeon Cho and Beom Sik Kang
on the other. The ATP lid in the structure
(a)
(b)
of PrrB ABD is not visible suggesting that
the ATP lid undergo a conformational
change (Figure 4c). The crystal structures
of DosS and DosT ABDs, which do not
have both F box and ATP lid motif, are
determined. The structures revealed that
they do not contain a sufficiently large
peptide region to form a complete ATP
lid motif, as found in other HKs. The
ATP binding sites are in a closed state
for ATP binding (Figure 4d, e). The DosS
and DosT ABD requires conformational
changes in the loop region to anchor ATP
and guide it to the DHp in the absence
of an ATP lid. Structural analyses and
FIGURE 5 I Arrangement of helices in DHp dimer. (a) Ribbon diagram of DHp domains found in
autophosphorylation assays of wild-type
Escherichia coli EnvZ (PDB ID 3ZRX). Two DHp domains are packed forming an antiparallel four helical
and mutant DosS kinase core suggest
bundle (left). The conserved histidine (H342) shown as stick are exposed (right). (b) The cartoon looking
down the four-helix bundle (α1 and α2 from one domain and α1’ and α2’ from another domain) and
that an interaction between the DHp
two ATP binding domains (CA and CA’) of the DHp domain dimer (left) and the arrangement of the four
domain and ABD is required, not only for
helices (right). The connection of the helices is portrayed by an arrow, and the linker between domains is
autophosphorylation, but also to trigger
depicted as a curve. Depending on loop handedness in the DHp dimer, the ATP binding domain is closer
to either the histidine on the same chain (cis) (top) or the histidine on the opposite chain (trans) (bottom)
the opening of the ATP binding site. Ionic
(Ashenberg et al., 2013).
interactions between Arg440 in DHp
domain and Glu537 in ABD are involved in the
activation step for ATP binding to DosS (Cho et
al., 2013).
posits that the handedness of a loop connecting two helices
DIMERIZATION AND HISTIDINE
PHOSPHOTRANSFERASE DOMAIN
DHp domain contains the phosphate-accepting histidine,
which is absolutely conserved in HKs and is the signature
motifs defining a HK. The histidine residue is the site for
autophosphorylation and subsequent transfer of the phosphoryl
group to cognate RRs (Dutta et al., 1999). Although there is no
structure for the DHp domains from M. tuberculosis HKs yet, one
can expect that the structures of the DHp domains based on
the structures of other HKs, such as DesK from Bacillus subtilis
(Albanesi et al., 2009) and HK853 from Thermotoga maritima
(Marina et al., 2005). DHp domains of DesK and HK853 show
sequence similarity to those of DosS (32% sequence identity)
and TcrY (36% sequence identity) representing each of the two
M. tuberculosis HK groups arranged by ABD, respectively. DHp
domain consists of two long α-helices (α1 and α2) that form an
antiparallel coiled-coil and it has conserved histidine residue on
helix α1 and highly solvent exposed. DHp domain participates in
dimerization of the HK through the association of two protomers
forming a four-helix bundle (Figure 5a).
HKs function as dimers in which usually one monomer
catalyzes phosphorylation of the histidine residue in the other
monomers. It is called “trans-autophosphorylation”. Some
HKs is known to catalyze its phosphorylation in “cis” mode.
The ABD of one monomer phosphorylates the histidine in the
same monomer. Based on structural considerations, one model
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of DHp domain at the base of the helical dimerization plays a
critical role (Ferris et al., 2012). Helix bundle loops determine
whether HKs autophosphorylate in cis or in trans (Ashenberg et
al., 2013). Depending on loop handedness in the DHp domain,
the ABD is closer to either the histidine on the same chain (cis)
or the histidine on the opposite chain (trans) (Ashenberg et al.,
2013) as shown HK853 (PDB ID 2C2A) (Marina et al., 2005) or
EnvZ (PDB ID 3ZRX) (Ferris et al., 2012), respectively (Figure 5b).
Structural studies for DHp domains in M. tuberculosis HKs would
be necessary for the understanding of the autophosphorylation
mechanism.
RESPONSE REGULATORS
Common RRs are consisted two major domains, a conserved
receiver domain and variable effector domain at its N- and
C-terminus, respectively (Figure 2h). Effector domains, which
are also called as output domains become activated upon
phosphorylation. In most cases, the effector domain is a DNA
binding domain regulating transcriptional initiation (Gao et
al., 2009; Stewart, 2010). The receiver domains of the RRs
are conserved and have three activities. One, catalysis of
phosphoryl transfer reaction from the phosphorylated histidine
of the DHp domain to the aspartate of receiver domain; Two,
autodephosphorylation of the aspartate; and three, regulation
of the activity of the effector domain in a phosphorylationdependent manner (West et al., 2001).
Since the majority of RRs are transcription regulators, their
Bio Design l Vol.3 l No.1 l Mar 30, 2015 © 2015 Bio Design
7
Two component signaling systems in Mycobacterium tuberculosis
(a)
(c)
(b)
(d)
(f)
HKs to their cognate RRs in TCS. The
receiver domain is also known as the
phosphorylation domain or regulatory
domain. A conserved aspartate residue
i n t h e re c e i v e r d o m a i n a c c e p t s a
phosphoryl group from a cognate HK
(Dutta et al., 1999). The receiver domain
structures of eight RRs (RegX3, PhoP,
NarL, PrrA, DosR, MtrA, PdtaR and an
orphan RR, GlnR) in M. tuberculosis
(e)
(g)
have been determined and display their
structural similarity. Amino acid sequence
alignment analyses show the remnant
RRs (TcrA, NarL, MprA, KdpE, TrcR,
and TcrX) also have a similar receiver
domain in their N-terminus linked to
their DNA binding domains (Shrivastava
et al., 2006; Cho et al., 2014). The
receiver domain has a conserved (β/α) 5
structure. Alternating β strands and α
helices fold into a five-stranded parallel
β-sheet surrounded by two α-helices
on one side and three α-helices on the
other side. Six functionally important
residues (three Asp/Glu, one Thr/Ser,
one Tyr/Phe, and one Lys) are conserved
for phosphorylation of the receiver
domain and the activation of the effector
domain (Stock et al., 1989; Bourret et
al., 1990). The two consecutive acidic
FIGURE 6 I Receiver domain structures of response regulators from Mycobacterium tuberculosis.
residues located in the β1-α1 loop
(a) Superimposition of the receiver domains of four response regulators, PhoP (PDB ID 3ROJ), NarL (PDB
coordinate a Mg2+ ion and the phosphorID 3EUL), PrrA (PDB ID 1YS6), and MtrA (PDB ID 3NHZ), which are presented in yellow, green, blue, and
magenta, respectively. (b) PhoP dimerization is through a two-fold symmetrical interaction of α4-β5-α5
accepting Asp are in the β3-α3 loop
motifs of the receiver domain. (c)~(e) The cartoon diagrams for the receiver domains of RegX3 (PDB
(Stock et al., 1993). The carboxylate
ID 2OQR), DosR (PDB ID 3C3W) and GlnR (PDB ID 4O1I) colored by secondary-structural elements
oxygen of the Asp residue executes
(α-helices, cyan; β-strands, magenta). (f) Comparison of the phosphorylation site in NarL (yellow) from M.
tuberculosis with the active site of histidinol phosphate phosphatase (slate) from E. coli (PDB ID 2FPW).
nucleophilic attack on the phosphorus
The phospho-aspartyl intermediate is formed at D57, and the catalytic Mg2+ is replaced by Ca2+ (a
atom for the phosphorylation of the
green sphere)(Schnell et al., 2008). (g) Proposed model of DosR autodephosphorylation for nucleophilic
receiver domain (Bourret, 2010).
substitution. A water molecule positioned and activated by the hydroxyl group of S83 performs a
nucleophilic in-line attack on the phosphorus, causing a planar PO3 transition state coordinated by
The conserved Lys residue located
conserved T82, K104, and the Mg2+. Residue numbers of histidinol phosphate phosphatase are in the
in the β5-α5 loop forms an ionic
parentheses (Cho et al., 2014).
interaction with the phosphoaspartate
in the phosphorylated RR (Lukat et
effector domains are DNA-binding domains. A significant fraction
al., 1991). Autodephosphorylation of the phosphoaspartate
of RRs have effector domains as enzymes. Other RRs have
is presumed to proceed through a similar mechanism to the
effector domains binding to RNA, ligands, or proteins regulating
autophosphorylation, but in reverse. A water molecule performs
cellular process (Galperin, 2010). Some RRs consist of an
nucleophilic in-line attack on the phosphorus. The conserved
isolated receiver domain alone. Those regulate target effectors
Thr/Ser, conserved Lys, and Mg2+ ion stabilize the planar PO3
through intermolecular interactions with functional proteins as
transition state.
shown in the chemotaxis protein CheY (Halkides et al., 2000).
PhoP has a canonical receiver domain structure in its
N-terminus (Menon et al., 2011). Like other receiver domains
RECEIVER DOMAINS
from the OmpR subfamily, it has a (βα)5 fold and the central fiveReceiver domains have well conserved structure and sequences,
stranded parallel β-sheet is surrounded by α1 and α5 helices on
suggesting a common mechanism to transfer signals from
one side and α2, α3, and α4 helices on the other side (Figure 6a).
8
Bio Design l Vol.3 l No.1 l Mar 30, 2015 © 2015 Bio Design
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Ha Yeon Cho and Beom Sik Kang
The β-strands are mainly composed of hydrophobic residues
and the α-helices are amphipathic. The side facing the β-sheet
is composed of hydrophobic residues while hydrophilic residues
are exposed to solvent. PhoP forms a dimer through the α4β5-α5 motif of the receiver domain. The dimer with this interface
has been proposed to be the active conformation (Figure 6b).
The receiver domains of PrrA (Nowak et al., 2006), MtrA (Barbieri
et al., 2010), PdtaR (Morth et al., 2004), and NarL (Schnell et al.,
2008) also have the expected (βα)5 topology, with five parallel
strands forming the hydrophobic core surrounded by two helices
(α1 and α5) on one side and three (α2–α4) on the other (Figure
6a). The phosphorylation site is conserved Asp located in the
loop at the C-terminal end of β3 strand and the site is covered
by a flexible loop between α3 and β3.
The N-terminal receiver domain of DosR contains an α/β fold
with a (βα)4 arrangement unlike the typical receiver domain.
The canonical secondary structure elements α4 and β5 appear
to be absent from the DosR structure (Figure 6c). Surprisingly,
the DosR α4 helix is positioned where α5 helix in the canonical
structures of other members the subfamily is located. The
catalytic Asp54 residue is located in the β3-α3 loop surrounded
by the conserved Asp8 and Asp9 residues from the β1-α1 loop
from the same subunit (Wisedchaisri et al., 2008). The monomer
of RegX3 has an apparently incomplete receiver domain
consisted of four-stranded parallel β-sheet and three α-helices.
Four-stranded β-sheet is flanked by α1 helix on one side of the
β-sheet and by α2 and α3 helices on the other side (Figure 6d)
(King-Scott et al., 2007). It seems to be a conformational change
in α4 helix of the receiver domain like DosR (Cho et al., 2014).
Inactive MtrA forms an extensive interface between the receiver
and effector domains over the α4-β5-α5 face of the regulatory
domain, the transactivation loop (α7-α8 loop) and the recognition
helix (α8) of the DNA-binding motif. Those functionally important
surfaces of each domain are sequestered (Friedland et al., 2007).
In the active state, the α4-β5-α5 face is proposed to mediate
dimerization of the regulatory domains, and the recognition helix
and transactivation loop play roles for interactions with DNA
and RNA polymerase, respectively. GlnR considered an orphan
RR because its cognate HK has not been identified. Its receiver
domain have canonical structure consisted of five β-strands and
five α-helices except that α1, α2, and α4 helices are partially
unwound (Figure 6e). The two GlnR monomers form a homodimer through their α4-β5-α5 and the interface is essential for its
physiological function (Lin et al., 2014).
EFFECTOR DOMAINS
Based on sequence similarity and structure of the effector
domain, RRs can be classified into subfamilies such as OmpR,
NarL, NtrC, LytR, ActR, and YesN-like subfamily (Stock et al.,
2000). More than 60% of RRs contain a DNA binding domain as
an effector domain and they are divided into subfamilies based
on the fold of DNA binding domain. Other subfamilies include
RRs containing RNA binding domains, a variety of enzymatic
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domains or protein-protein interaction domains (Galperin, 2006;
Gao et al., 2007). All RRs in M. tuberculosis have a DNA binding
domain except PdtaR, which contains a RNA binding domain
(Morth et al., 2004). DNA binding domains of M. tuberculosis
RRs can be grouped into two groups, NarL and OmpR families,
which have Helix-turn-helix (HTH) motif and winged HTH (wHTH)
motif, respectively. RRs containing wHTH motif are SenX3, TcrA,
PhoP, PrrA, MprA, KdpE, TrcR, MtrA, and TcrX, and they belong
to OmpR-like subfamily (Martínez-Hackert et al., 1997). NarL and
DosR have HTH motif and belong to NarL-like subfamily (Baikalov
et al., 1996).
HTH motifs are found in all DNA binding proteins regulating
gene expression. The motif consists of about twenty residues
and is characterized by two α-helices. They are joined by a short
turn and make close contacts with the DNA. The second α-helix
of the motif fits into the major groove of the DNA with specific
interactions between the side chains and the exposed bases,
while the first α-helix helps to stabilize the position of the second
α-helix. The wHTH motif consists of three α-helices (H1, H2, H3)
and three- or four-stranded β-sheet (wing). The H2-H3 region
forms a HTH motif and the DNA recognition helix (H3) makes
sequence-specific DNA contacts at the major groove, while the
wing makes DNA contacts at the minor groove or the backbone
of DNA. Many proteins with wHTH motif present an exposed
hydrophobic patch to mediate protein-protein interactions
(Martínez-Hackert et al., 1997).
M. tuberculosis PhoP, which belongs to the OmpR subfamily,
the largest subfamily of RRs (Wang et al., 2007) has the typical
structure of the wHTH motif in the C-terminal effector domain.
The domain starts with a four-stranded antiparallel β-sheet,
followed by three α-helices and a β-hairpin structure (Figure
7a). In solution it exists predominantly as a monomer (Menon
et al., 2011). The effector domains of PrrA (Nowak et al., 2006),
RegX3 (King-Scott et al., 2007) and MtrA (Friedland et al., 2007)
(Figure 7b~d, respectively), another OmpR family members
have also a winged helix fold composed of a four-stranded
antiparallel β-sheet followed by a three-helix bundle and a
C-terminal β-hairpin. The DNA recognition helix (α8) of RegX3
is fully exposed to the solvent as the case for the OmpR family
members DrrB (Robinson et al., 2003) and DrrD (Buckler et al.,
2002).
DosR as a member of the NarL subfamily has C-terminal
domain containing four α-helices (α7, α8, α9 and α10) on the
basis of sequence alignment of the protein family (Wisedchaisri
et al., 2005). The crystal structures of the DosR effector domain
and its complex with DNA confirm that DosR is a member of
the NarL subfamily of RRs. A dimer of DosR effector domain
is the functional unit for DNA binding where the residues in
the α10 helix form the dimerization interface (Figure 7e). Two
DNA-binding dimers gather to form a tetramer through α7 and
α8 helices of the effector domain. The crystal structure of fulllength unphosphorylated DosR reveals a novel topology of the
N-terminal receiver domain and a unique conformation for the
Bio Design l Vol.3 l No.1 l Mar 30, 2015 © 2015 Bio Design
9
Two component signaling systems in Mycobacterium tuberculosis
(a)
(b)
(e)
(c)
(d)
(f)
(g)
FIGURE 7 I Effector domain structures of response regulators from Mycobacterium tuberculosis. (a)~(d) Effector domains of PhoP (PDB ID 2PMU) (a), PrrA
(PDB ID 1YS6) (b), RegX3 (PDB ID 2OQR) (c), and MtrA (PDB ID 2GWR) (d), which have a wHTH motif. (e) A dimer of DosR effector domains binding DNA. The key
residues for the interaction (T198, V202, and T205) for the dimerization are on the helix α10, and a9 helices are inserted in the major groove (Wisedchaisri et al.,
2005). (f) Superposition of the three different structures of DosR C-terminal domain are superimposed; the domains from the full-length protein (green), a complex
with DNA (orange), and in crystal form II (cyan) (20). (g) The HTH motif of PdtaR effector domain (PDB ID 1S8N). Ribbon diagram (left) and the superimposition
(rignt) of the motif of PdtaR (red) on that of ANTAR domains of AmiR (Cyan) from Pseudomonas aeruginosa. The side chains believed to be involved in tertiary
structure organization are shown in ball-and-stick (Morth et al., 2004).
C-terminal effector domain. The effector domain in the full-length
DosR structure shows a novel position of α10 helix, which allows
Gln199 to interact with the catalytic Asp54 residue of the receiver
domain, while the structure of the DosR effector domain alone
displays an unstructured conformation for α10 helix, indicating
considerable flexibility (Figure 7f). Activation of DosR induced
by phosphorylation is though conformation changes by a helix
rearrangement (Wisedchaisri et al., 2008).
PdtaR contains an RNA binding domain only found in M.
tuberculosis (Morth et al., 2004). The C-terminal effector domain
of PdtaR is structurally homologous to the ANTAR domain of
AmiR from Pseudomonas aeruginosa. It is known to be involved
in transcriptional antitermination (Shu et al., 2002). It has a helical
bundle made of three α-helices and the geometrical arrangement
of helices in the ANTAR domain is highly conserved between
the domains (Figure 7g). It seems likely that the binding mode of
10
Bio Design l Vol.3 l No.1 l Mar 30, 2015 © 2015 Bio Design
PdtaR is similar to that of NarL to DNA.
AUTODEPHOSPHORYLATION
Phosphorylation on the receiver domain facilitates a conformational alteration affecting RR to work as a transcriptional
activator. Genes activated in this manner tend to remain active
until the RR is deactivated by dephosphorylation (West et al.,
2001). In many TCS, the primary route of RR dephosphorylation
is through the phosphatase activity of other proteins. RR is
also dephosphorylated by its cognate HK or by itself called
“autodephosphorylation”. The active site in the receiver
domain of RRs is similar to that of members of the haloacid
dehalogenase (HAD) family, a family of phosphatases, because it
also forms a phospho-aspartyl intermediate (Seifried et al., 2013;
Rangarajan et al., 2006). An aspartic acid acting as the catalytic
acid/base in HAD enzymes is substituted by an arginine in
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Ha Yeon Cho and Beom Sik Kang
NarL (Figure 6f). It is unlikely to participate in the step of proton
abstraction. This replacement may prolong the phospho-aspartyl
state.
Among RRs in M. tuberculosis, NarL has high sequence
similarity to DosR, and autophosphorylated DosS can
transfers its phosphate group not only to DosR but also to
NarL. Phosphorylated DosR is more rapidly dephosphorylated
than phosphorylated NarL. DosR and NarL differ with respect
to the amino acids at positions T + 1 and T + 2 around the
phosphorylation sites in the N-terminal receiver domain; DosR
has Ser83 and Tyr84, whereas NarL has Ala90 and His91. A
DosR Ser83Ala mutant shows prolonged phosphorylation.
Structural comparison with a histidinol phosphate phosphatase
(Figure 6g) suggests that the Ser83 at the T + 1 position provides
space for a water molecule and its hydroxyl group is involved
in the activation of the water molecule for the triggering of
autodephosphorylation (Cho et al., 2014).
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CONCLUDING REMARKS
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Here, we summarize the current structural studies on the TCS
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RRs are structurally very preserved. PAS and GAF domains
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development.
ACKNOWLEDGEMENTS
This work was supported by a National Research Foundation of Korea
(NRF) grant funded by the Korean government (MEST)(2011-0015987).
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Accepted: Mar 3, 2015
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