Identical phosphatase mechanisms achieved through distinct modes of binding phosphoprotein substrate.
ABSTRACT Two-component signal transduction systems are widespread in prokaryotes and control numerous cellular processes. Extensive investigation of sensor kinase and response regulator proteins from many two-component systems has established conserved sequence, structural, and mechanistic features within each family. In contrast, the phosphatases which catalyze hydrolysis of the response regulator phosphoryl group to terminate signal transduction are poorly understood. Here we present structural and functional characterization of a representative of the CheC/CheX/FliY phosphatase family. The X-ray crystal structure of Borrelia burgdorferi CheX complexed with its CheY3 substrate and the phosphoryl analogue reveals a binding orientation between a response regulator and an auxiliary protein different from that shared by every previously characterized example. The surface of CheY3 containing the phosphoryl group interacts directly with a long helix of CheX which bears the conserved (E - X(2) - N) motif. Conserved CheX residues Glu96 and Asn99, separated by a single helical turn, insert into the CheY3 active site. Structural and functional data indicate that CheX Asn99 and CheY3 Thr81 orient a water molecule for hydrolytic attack. The catalytic residues of the CheX.CheY3 complex are virtually superimposable on those of the Escherichia coli CheZ phosphatase complexed with CheY, even though the active site helices of CheX and CheZ are oriented nearly perpendicular to one other. Thus, evolution has found two structural solutions to achieve the same catalytic mechanism through different helical spacing and side chain lengths of the conserved acid/amide residues in CheX and CheZ.
- SourceAvailable from: Victoria Robinson
Article: Two-component signal transduction.[show abstract] [hide abstract]
ABSTRACT: Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein. The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the regulatory domain that results in activation of an associated domain that effects the response. The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems. The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained. Thus detailed analyses of a relatively small number of representative proteins provide a foundation for understanding this large family of signaling proteins.Annual Review of Biochemistry 02/2000; 69:183-215. · 27.68 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The relevance toward virulence of a variety of two-component signal transduction systems is reviewed for 16 pathogenic bacteria, together with the wide array of environmental signals or conditions that have been implicated in their regulation. A series of issues is raised, concerning the need to understand the environmental cues that determine their regulation in the infected host and in the environment outside the laboratory, which shall contribute toward the bridging of bacterial pathogenesis and microbial ecology.Microbial Ecology 03/2006; 51(2):166-76. · 3.28 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: All living organisms, whether they be single- or multi-cellular, actively interact with surrounding environments and modulate their physiological status to maintain cellular homeostasis. ...Eukaryotic Cell 11/2008; 7(12):2017-36. · 3.59 Impact Factor
Identical phosphatase mechanisms achieved through
distinct modes of binding phosphoprotein substrate
Y. Pazya, M. A. Motalebb,c, M. T. Guarnierid, N. W. Charonc, R. Zhaod, and R. E. Silversmitha,1
aDepartment of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599;
Carolina University, Greenville, NC 27834;
bDepartment of Microbiology and Immunology, East
cDepartment of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV 26506;
dDepartment of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO 80045
Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved November 18, 2009 (received for review October 1, 2009)
Two-component signal transduction systems are widespread in
prokaryotes and control numerous cellular processes. Extensive in-
vestigation of sensor kinase and response regulator proteins from
many two-component systems has established conserved se-
quence, structural, and mechanistic features within each family.
In contrast, the phosphatases which catalyze hydrolysis of the re-
sponse regulator phosphoryl group to terminate signal transduc-
tion are poorly understood. Here we present structural and
functional characterization of a representative of the CheC/CheX/
FliY phosphatase family. The X-ray crystal structure of Borrelia
burgdorferi CheX complexed with its CheY3 substrate and the
phosphoryl analogue BeF−
a response regulator and an auxiliary protein different from that
shared by every previously characterized example. The surface
of CheY3 containing the phosphoryl group interacts directly with
a long helix of CheX which bears the conserved (E − X2− N) motif.
Conserved CheX residues Glu96 and Asn99, separated by a single
helical turn, insert into the CheY3 active site. Structural and func-
tional data indicate that CheX Asn99 and CheY3 Thr81 orient a
water molecule for hydrolytic attack. The catalytic residues of
the CheX · CheY3 complex are virtually superimposable on those
of the Escherichia coli CheZ phosphatase complexed with CheY,
even though the active site helices of CheX and CheZ are oriented
nearly perpendicular to one other. Thus, evolution has found two
structural solutions to achieve the same catalytic mechanism
through different helical spacing and side chain lengths of the
conserved acid/amide residues in CheX and CheZ.
3reveals a binding orientation between
CheX ∣ CheY ∣ two-component systems ∣ dephosphorylation ∣
development) in prokaryotes, fungi, and plants (1–4). To date,
genes for more than 50,000 proteins that function in two-
component systems have been identified by genome sequencing
projects (www.p2cs.org). In two-component systems, a sensor
kinase autophosphorylates on a histidyl residue. The phosphoryl
group is then transferred to an aspartyl residue on the receiver
domain of a cytoplasmic response regulator protein, which acti-
vates the response regulator to execute a cellular response to a
stimulus. Deactivation of the response regulator occurs by
hydrolysis of the phosphoryl group, sometimes via an auxiliary
In contrast to sensor kinases and response regulators [see
refs. (5–7) for recent reviews], the mechanisms by which auxiliary
phosphatases operate are poorly characterized. At least five
distinct families of response regulator phosphatases have been
identified to date: CheC/CheX/FliY (8), CheZ (9), Rap (10), sen-
sor kinases (11, 12), and Spo0E (13). However, the active site has
been definitively identified and a reaction mechanism established
for only the CheZ class of phosphatases. In principle, structural,
mechanistic, or amino acid sequence characteristics which are
shared between families of response regulator phosphatases
could be used to predict which proteins from genomic databases
wo-component signal transduction systems control diverse cel-
lular processes (ranging from pathogenesis to morphological
possess phosphatase activity toward response regulators. Here we
use structural and enzymatic methods to investigate a represen-
tative of the CheC/CheX/FliY class of phosphatases.
The CheC/CheX/FliY family spans diverse phyla within bac-
teria and archaea (8, 14). X-ray crystal structures of Thermotoga
maritima chemotaxis proteins CheX and CheC (15) reveal similar
folds which reflect an internal sequence pseudosymmetry (16). A
conserved (E-X2-N) motif is present twice in CheC due to the
pseudosymmetry, but CheX contains only the second copy of
the motif. For each motif, the conserved glutamate and aspara-
gine are one helical turn apart on a solvent exposed alpha helix.
Mutagenesis studies (15, 17, 18) suggest that the (E-X2-N) motif
mediates catalysis. The (E-X2-N) motifs in CheC and CheX ap-
pear comparable to the structurally unrelated CheZ phosphatase,
whose active site acid and amide residues Asp143 and Gln147 are
also separated by a single helical turn (9). However, attempts to
superimpose a catalytic motif of CheC onto its counterpart in
CheZ suggested that the mode of interaction of the CheC/
CheX/FliY phosphatases with CheY differs from CheZ (15).
Here, we present the X-ray crystal structure of a complex con-
taining Borrelia burgdorferi CheX and its CheY3 substrate bound
to the phosphoryl analogue BeF−
operate in the two-component system which mediates chemotaxis
in B. burgdorferi (19–21), the causative agent of Lyme disease.
The structure reveals a unique mode of binding between a re-
sponse regulator and an auxiliary protein, but a catalytic mecha-
nism which is virtually identical to that used by the structurally
unrelated CheZ, providing a striking example of convergent
evolution. The data also suggest a possible CheX regulatory
mechanism through dissociation of the CheX homodimer.
3and Mg2þ. CheX and CheY3
Overall Structure of CheX · CheY3 · BeF−
ture of B. burgdorferi CheX · CheY3 · BeF−
mined at a resolution of 1.96 Å (Table S1). The asymmetric
unit contained one molecule each of CheX and CheY3. The
2Fo-Fcelectron density map displayed clear density for residues
4–158 of CheX (160 residues total) with the exception of weak
density for several short stretches (residues 33–40, 109–113,
and 125–136), all ofwhich are distant from the interaction surface
with CheY3. There was clear electron density for residues 13–146
of CheY3 (146 residues total), as well as the BeF−
bound to the CheY active site. Figure 1A shows the asymmetric
unit and illustrates that the interaction between the proteins
brings the conserved residues Glu96 and Asn99 of CheX proxi-
mal to the BeF−
3· Mg2þ. The crystal struc-
3· Mg2þwas deter-
3moiety in CheY3.
Author contributions: Y.P., M.A.M., N.W.C., and R.E.S. designed research; Y.P., M.A.M., and
R.E.S. performed research; N.W.C. contributed new reagents/analytic tools; Y.P., M.A.M.,
M.T.G., R.Z., and R.E.S. analyzed data; and M.A.M. and R.E.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
1924–1929 ∣ PNAS ∣ February 2, 2010 ∣ vol. 107 ∣ no. 5www.pnas.org/cgi/doi/10.1073/pnas.0911185107
B. burgdorferi CheX Is Not Present As a Homodimer in the Cocrystal.
Superposition of B. burgdorferi CheY3 · BeF−
CheY · BeF−
0.8 Å for Cα atoms within secondary structure elements), active
site geometries, and rotameric positions of residues which change
upon activation, indicating that CheY3 was in a fully active con-
formation in the cocrystal (Fig. S1). CheY3 residues 13–26, part
of an extended amino terminus absent in other CheYs, formed an
ordered random coil which did not interact with other residues in
CheY3, but made crystal contacts with a CheX chain from a
neighboring asymmetric unit. Overlay of the CheX chains from
CheX · CheY3 · BeF−
showed good backbone alignment for a majority of the secondary
structure elements (rmsd ¼ 0.9 Å for Cα atoms). However,
several regions showed poor alignment. Notably, a continuous
in poor alignment with T. maritima CheX. These residues corre-
spond to the β10-βx0loop, βx0, and the βx0-β20loop in T. maritima
CheX2, which comprise a large portion of the intradimer surface.
There was also weak electron density for residues 37–40 of
B. burgdorferi CheX, which could not be modeled into the struc-
ture (Fig. 1B). These residues correspond to the βx strand in
T. maritima CheX, which interacts directly with βx0. Therefore
the regions of disorder in B. burgdorferi CheX are localized
to the regions which mediate intersubunit interactions in the
T. maritima CheX2structure.
T. maritima CheX forms a dimer in both the crystal and solu-
tion (15). Although the asymmetric unit of the CheX · CheY3 ·
homodimer could potentially be formed from two CheX chains
contributed from different asymmetric units. However, genera-
tion of all of the neighboring asymmetric units did not yield a
CheX monomer in a position correlating to the second subunit
3· Mg2þand E. coli
3· Mg2þrevealed highly similar backbones (rmsd ¼
3· Mg2þand T. maritima CheX (Fig. 1B)
3· Mg2þstructure contained only one chain of CheX, a
in T. maritima CheX2, nor was a dimer with an alternate surface
of interaction implicated. The mobility of CheX · CheY3 ·
sistent with a complex which contained one molecule each of
CheX and CheY3 (Fig. S2).
The observation that CheX is not present as a homodimer in
the CheX · CheY3 · BeF−
cause gel filtration analysis indicated that purified B. burgdorferi
CheX is dimeric in solution (20). Because gel filtration can give
misleading results for some proteins, we used a combination of
Rayleigh light scattering and refractometry to determine the as-
sociative state of CheX. This analysis gave a polydispersity index
of 1.000 ? 0.003, indicating homodispersity of the CheX sample,
and a molecular weight of 36,980 (cf. CheX2theoretical molecu-
lar weight of 37,760), confirming CheX2 dimer formation.
Therefore, it appears that binding CheY3 · BeF−
results in dissociation of the CheX2dimer. The disorder in
the portions of B. burgdorferi CheX which correspond to the
intradimer surface in T. maritima CheX2are likely due to the loss
of stabilizing interchain interactions within the CheX2dimer.
3· Mg2þon an analytical gel filtration column was also con-
3· Mg2þcomplex was unexpected be-
3· Mg2þto CheX
Novel Orientation of CheY3 and CheX Within CheX · CheY3 ·
CheY3 is oriented relative to CheX so that the α1 helix in CheY3
is essentially perpendicular to the α1′ helix in CheX which bears
the conserved (E-X2-N) sequence motif (Figs. 1A, 2). Residues
throughout the entire length of CheX α10, as well as the CheX
α3-α10loop and the β10strand, interact with residues on all of
the CheY3 βα loops except β2α2, as well as the N-terminal
two turns of α1 (Fig. S3). The area of the interaction surface be-
tween CheX and CheY3 is 896 Å2and contains five interchain
hydrogen bonds and one salt bridge interspersed with patches
of hydrophobic interactions (Fig. S3). Table S2 lists intermolecu-
lar interactions within the CheX · CheY3 · BeF−
The relative orientation of CheY3 and CheX in the cocrystal
structure contrasts that of the three available high resolution
structures of a complex containing a receiver domain and a pro-
tein which mediates phosphoryl group chemistry (Fig. 2). These
structures are E. coli CheY complexed with the CheZ phospha-
tase (9) and two complexes between a receiver domain and a his-
tidyl phosphotransferase (22, 23), a protein which mediates
phosphotransfer between its own histidyl residue and the receiver
domain aspartate. In all three structures, a surface comprised of
one or two near-parallel alpha helices of the partner protein
interacts with the active site region of the receiver domain but
the receiver domains are tilted so that the α1 helix is at an angle
of about 30° with respect to the dominant interacting helix on
CheX · CheY3 · BeF−
rotated about 40° around an internal axis roughly central and
parallel to their β-strands relative to CheY3 (Fig. 2).
3· Mg2þ: In the CheX · CheY3 · BeF−
3· Mg2þ. The receiver domains are also
The Interaction Surface Between CheY3 and CheX Includes the Cata-
lytic Site. Inspection of the CheY3 active site region (Fig. 3A) re-
veals that the two strictly conserved CheX residues, Glu96 and
Asn99, which reside one helical turn apart on CheX α10, are ad-
jacent to the CheY3 active site to form a region with an extensive
network of hydrogen bonds. CheX Glu96 OE2 forms a salt bridge
with conserved residue CheY3 Lys129 NZ as well as a hydrogen
bond with CheY3 Thr37 OG1. The CheX Asn99 side chain is
pointed toward the BeF−
ND2 forms a hydrogen bond with an ordered water molecule
(water B162) which is located in a position close to that expected
for in-line attack of the phosphoryl group—3.4 Å from the Be
atom and <1 Å from a direct in-line position. Water B162 forms
an additional hydrogen bond with CheY3 Thr81 OG, implicating
roles for both CheXAsn99 and CheY3 Thr81 in orienting a water
3moiety on CheY3 so that Asn99
sentation of the asymmetric unit. CheX is cyan and CheY3 is green. CheY3
Asp79 (orange), BeF−
(red, Lower) are in stick representation. Mg2þis a magenta sphere. (B).Over-
lay of B. burgdorferi CheX (cyan) and T. maritima CheX chain B (pdb 1XKO,
tan). Regions of B. burgdorferi CheX with high disorder (Cα thermal B-factors
>70) are blue. B. burgdorferi CheX residues 37–40 are sketched as blue
dots. T. maritima CheX residues corresponding to the disordered regions
of B. burgdorferi CheX are red.
Overall topology of CheX · CheY3 · BeF−
3· Mg2þ. (A) Ribbon repre-
3(purple), CheX Glu96 (red, Upper), and CheX Asn99
Pazy et al. PNAS
February 2, 2010
molecule for nucleophilic attack in CheX-mediated catalysis of
the dephosphorylation reaction.
The active site configuration observed in the CheX · CheY3 ·
E. coli CheZ · CheY · BeF−
CheZ (Asp143) forms a salt bridge with CheY Lys109 and an
amide residue on the next turn of the CheZ helix (Gln147) inserts
into the CheY active site. However, in the CheZ · CheY ·
sign density to ordered water molecules and the residue analo-
gous to CheY3 Thr81, CheY Asn59, was not implicated as
catalytically important based on the structure. However, the anal-
ogous residue of phosphatases within the haloacid dehalogenase
family, which possess similar folds and active site geometries as
response regulators, is conserved as an aspartate and has been
proposed to act as a general base toward the nucleophilic water
for hydrolysis of the aspartyl-phosphate intermediate (24).
3· Mg2þcomplex is directly comparable to that observed in
3· Mg2þwhere an acid residue on
3· Mg2þstructure, there was not sufficient resolution to as-
Enzymatic and Genetic Assays Support Mechanistic Roles for CheX and
CheY3 Residues Implicated by the Cocrystal Structure. Potential roles
for CheX Glu96, CheX Asn99 and CheY3 Thr81 in the CheX-
stimulated dephosphorylation of CheY3 were assessed by enzyme
kinetic analysis (Fig. 3B). For wild-type CheY3 in the absence
of CheX, the rate of inorganic phosphate (Pi) release (using
phosphoimidazole as a phosphodonor) reflects CheY3 autode-
phosphorylation and displayed a rate constant (7.8 ? 0.1×
10−4s−1), similarto measurements
½32P?CheY3-P (20). Increasing concentrations of wild-type CheX
enhanced the Pirelease rate in a linear fashion and gave an
obtained by using
activity of 3.3 ? 0.3 × 10−2μM Pi∕s · μM CheX. In contrast,
3.3 ? 1.4% and 9.7 ? 4.4% of wild-type CheX activity, respec-
tively, consistent with catalytic and/or binding roles for both
CheX residues. CheY3 T81A exhibited an autodephosphoryla-
tion rate constant which was decreased about threefold compared
to wild-type CheY3. Moreover, titration of CheY3 T81A with
of YPD1 (a histidyl phosphotransferase) and SLN1-R1 (a receiver domain)
within YPD1 · SLN1-R1 · BeF−
Mg2þ(Upper), CheX is gray except for α10, which is cyan. CheY3 is green
except α1 is blue and α5 is red. For YPD1 · SLN1-R1 · BeF−
YPD1 is gray except for αC (teal). SLN1-R1 is pale yellow except α1 is blue
and α5 is red.
Comparison of the relative orientation of CheX and CheY3 with that
3· Mg2þ(pdb 2R25). For CheX · CheY3 · BeF−
(A)Close-upview of the active site. Residues are green for CheY3 and cyan for
CheX. The ordered water molecule (Wat B162) is a blue sphere and Mg2þis a
light green sphere. Hydrogen bonds revealed by the structure are repre-
sented by black-dashed lines. The red-dashed line between Wat B162 and
the beryllium atom (distance of 3.4 Å) is nearly collinear with the bond con-
necting the beryllium atom and Asp79 OD1. (B) CheX phosphatase activities.
The CheY3 concentration is 15 μM. Reactions contained wild-type CheX/wild-
type CheY3 (closed squares), CheX 96EA/wild-type CheY3 (closed triangles),
CheX 99NA/wild-type CheY3 (closed diamonds), or wild-type CheX/ CheY3
81TA (closed circles). Rates measured in the absence of CheY3 for wild-type
CheX (open squares), CheX 96EA (open triangles) and CheX 99NA (open dia-
monds) reflects nonspecific phosphatase activity due to trace contaminants,
and was subtracted out. (C) Schematic model for transition state stabilization
in CheX -catalyzed dephosphorylation of CheY3. The bipyramidal transition
state iscoloredblack with dashed lines representing partial bonds. Stabilizing
interactions are represented with red-dashed lines with the assumption that
interactions observed in CheX · CheY3 · BeF−
state. One of several possible hydrogen bonding arrangements between
CheY3 T81 or CheX N99 and the water is shown. Residues from CheY3 (green)
and CheX (cyan) are indicated.
Active site structure and kinetic characterization of CheX and CheY3.
3· Mg2þpersist in the transition
www.pnas.org/cgi/doi/10.1073/pnas.0911185107Pazy et al.
wild-type CheX indicated only ∼1–2% of the CheX sensitivity of
wild-type CheY3. Thus, Thr81 plays a modest role in CheY3
autodephosphorylation and a major role in CheX-mediated
dephosphorylation. Taken together (see Fig. 3C for a model of
proposed transition state and stabilizing interactions), the struc-
tural and kinetic data indicate that the CheX Asn99 and CheY3
Thr81 side chains participate directly in catalysis by orienting the
nucleophilic water. It is assumed that the proton lost by the
attacking water in the reaction is picked up by a solvent water
molecule. The primary role of Glu96 appears to be in binding
the CheY3-P substrate, based on its involvement in both a hydro-
gen bond and salt bridge with CheY3 residues (Figure 3A), and
consistent with diminished CheY binding affinity of CheC mu-
tants containing substitutions at the analogous acid residues (17).
An independent experiment measuring wild-type CheX activ-
ity as a function of wild-type CheY3 concentration demonstrated
that the CheX activity determined under the conditions used for
Fig. 3B represented a maximal velocity. Thus, CheX enhances the
rate of dephosphorylation of CheY3 by a factor of ≈40
(3.3 × 10−2s−1∕7.8 × 10−4s−1), similar to the enhancement of
CheY dephosphorylation by CheZ of ≈100-fold (25).
Cells containing plasmids with the cheX E96A and cheX N99A
mutations gave the genetic phenotype predicted for defective
phosphatase activity in a complementation assay. Cells with a dis-
abled cheX gene exhibit a constant flexing swimming phenotype
due to increased levels of phosphorylated CheY3 (20). A vector
expressing a wild-type copy of the cheX gene restored the swim-
ming behavior of the mutant B. burgdorferi cells to wild-type
behavior (a combination of run, pause, reverse and flex events),
but cells which were transformed with plasmids containing cheX
E96A or cheX N99A failed to complement the cheX mutant
phenotype and constitutively flexed.
CheX and CheZ Catalytic Mechanisms Are Virtually Identical Despite
DistinctInteraction Modes.The preceding structural and functional
analysis demonstrates that two conserved residues on CheX-
Glu96 and Asn99- interact directly with the active site of CheY3
and play important roles in catalysis. This is directly comparable
to the E. coli chemotaxis phosphatase CheZ where conserved re-
sidues Asp143 and Gln147 insert into the CheY active site and
possess binding and/or catalytic roles (9). Superposition of the
CheY and CheY3 molecules in the CheX · CheY3 · BeF−
Mg2þand CheZ · CheY · BeF−
veals that whereas the CheX and CheZ structures overlay poorly
(Fig. 4A), the catalytic residues assume virtually identical loca-
tions with respect to the CheY/CheY3 active site geometry
(Fig. 4B). Moreover, the functional atoms within the residues
(the carboxylic acid oxygen atom or the amide nitrogen atom)
are themselves essentially superimposable between CheX and
CheZ. Thus, CheX and CheZ mediate dephosphosphorylation
of their partner CheYs by essentially identical mechanisms. It
is striking that, despite the near superimposability of the active
sites, the alpha helices bearing the catalytic residues in CheX
and CheZ are oriented nearly perpendicular with respect to each
other (Fig. 4B), a manifestation of the distinct overall orientation
of CheY3 relative to CheX discussed above.
How can the two distinct orientations of an alpha helix ob-
served with CheX and CheZ result in near identical placement
of two catalytically essential residues which are, in both proteins,
separated by a single turn of an alpha helix? A conspicuous dif-
ference between the catalytic pair in CheX (Glu96 and Asn99)
and CheZ (Asp143 and Gln147) is their separation by two other
residues in CheX and three residues in CheZ. As a result, the Cα
atoms of the two catalytic residues are separated by different dis-
tances in CheX (4.79 Å) and CheZ (6.25 Å) and the side chains
project from the helices at different angles. Hence, a different
orientation of the helix bearing these residues is necessary to
get the functional atoms of side chains in the same approximate
3· Mg2þcocrystal structures re-
positions in space, and a near perpendicular backbone orienta-
tion achieves this goal. Finally, the positions of the functional
atoms are fine-tuned by the different lengths of the side chains.
The longer side chain of the acid residue (Glu96) and shorter side
chain of the amide residue (Asn99) in CheX versus CheZ
(Asp143 and Gln147, respectively) places the functional atoms
in nearly identical positions (Fig. 4B).
Identical Catalytic Strategies Achieved Through Distinct Binding
Modes.CheX and the structurally unrelated CheZ both use a pair
of functional groups—an acid and an amide—as a bidentate
prong which inserts into their respective CheY active sites. Dif-
ferences in residue spacing on the primary sequence and side
chain length are compensated for by disparate binding orienta-
tions of CheX and CheZ with the end result of the acid and amide
groups positioned in virtually identical locations within the CheY
active site. Examples of such “mechanistic” convergent evolution
amongst enzyme active sites have been documented (26, 27).
However, the mechanistic convergence demonstrated by CheX
and CheZ is distinct from that of conventional enzymes which
catalyze reactions of small molecules. CheX and CheZ do not
possess a typical active site pocket, but instead bind a protein sub-
strate and contribute additional functional groups to enhance the
activity of a preexisting active site. Thus, CheX and CheZ have
evolved both the ability to bind their protein substrate with ap-
preciable affinity [Fig. S2, (25)] and to provide the appropriate
functional groups in the right locations at the protein interface
to mediate catalysis. CheX and CheZ achieve these dual abilities
quite differently. CheX contains one protein surface which
mediates both binding and catalysis whereas CheZ separates
the binding and catalytic functions into two separate domains (9).
Could the Conserved Catalytic Mechanism Used by CheX and CheZ Be
Utilized by Structurally Different Classes of Response Regulator Phos-
phatases? The identical geometries of the active sites in
CheX · CheY3 and CheZ · CheY raises the possibility that the
conserved catalytic strategy represents a universal solution for
stimulation of response regulator dephosphorylation. The Spo0E
family of response-regulator phosphatases form short helical
hairpins with a conserved (-SQELD-) motif (28), containing an
exposed Asp/Gln pair. However, whereas the identities of the
Asp/Gln pair match CheZ, they are in reverse order and their
separation by two residues is reminiscent of CheX. Mutation
representation of B. burgdorferi CheX · CheY3 · BeF−
CheZ · CheY · BeF−
and CheY (light green). CheX is cyan and CheZ is orange. CheX Glu96 (Left)
and Asn99 (Right) are in cyan sticks. CheZ Asp143 (Left) and Gln147 (Right)
are in orange sticks. The BeF−
within the indicated square from Panel (A). Select active site groups from
B. burgdorferi CheY3 (green) or E. coli CheY (light green) are shown. The
residues are sticks and the divalent cations (Mg2þfor CheY3 and Mn2þfor
CheY) are spheres.
Two structural solutions achieve the same catalytic end. (A) Ribbon
3· Mg2þand E. coli
3· Mg2þ(pdb 1KMI) with superposition of CheY3 (green)
3moiety is in purple sticks. (B) Close-up of region
Pazy et al.PNAS
February 2, 2010
of the conserved Spo0E aspartate has a greater detrimental affect
on catalytic activity than mutation of the glutamine, leading to the
proposal that the aspartate interacts with the nucleophilic water
(29). It is possible that the Spo0E helix binds to its substrate with
the same directionality as observed for both CheX and CheZ.
Based on the separation of the glutamine and aspartate by
two residues, it is predicted that the helix bearing the catalytic
dyad would be oriented similarly to the CheX helix. This inter-
action would put the Spo0E glutamine in the position to form a
hydrogen bond with the response regulator lysine and the Spo0E
aspartate in the position to interact with the attacking water
molecule. This model, if shown to be correct, would suggest that
a common fundamental catalytic strategy could be modified by
adjusting the chemical properties of similarly located residues.
Implications for Interaction of CheY with CheC. The mode of binding
between phosphorylated CheYand CheX described here should
be applicable to CheC and FliY, two other CheY phosphatases
which are closely related to CheX. CheC has a similar topology as
CheX but is monomeric with two active site motifs—one on α10
(as in CheX) and one on α1 (15, 17). We generated a reasonable
model for a CheC · ðCheY · BeF−
laying a stretch of ten residues centered around the conserved
Glu/Asn for each catalytic motif in CheC with the homolo-
gous region in CheX. CheC and the deamidase, CheD, form a
heterodimer which mediates reciprocal regulation of enzymatic
activities (30), but the mechanism by which CheD activates CheC
is not known. Superposition of CheC from T. maritima
CheC · CheD (pdb 2F9Z) with CheC within the modeled
CheC·ðCheY · BeF−
closest, >10 Å removed from CheY (Fig. S4), inconsistent with
direct interaction between CheD and CheY in a ternary complex.
Therefore CheD does not appear to act to expand the surface of
interaction with CheY, and may instead act as an allosteric effec-
tor of CheC.
3Þ2complex (Fig. S4) by over-
3Þ2complex reveals that CheD is, at its
CheY3-Dependent Dissociation of CheX2: Possible Mechanism and
Function. The mechanism by which binding CheY3 · BeF−
presumably CheY3–P) induces the dissociation of the stable
CheX2dimer remains to be determined. The apparent limited
degree of overlap between CheY3 and CheX interaction surfaces
on the CheX monomer (Fig. S5) suggests that CheY3 · BeF−
might bind directly to the intact CheX2dimer which could some-
how induce dissociation of CheX2. Alternatively, the CheX2
dimer could be in equilibrium with a small amount of monomer.
CheY3 · BeF−
"pulling" the dimer/monomer equilibrium toward the monomeric
state. The functional significance of dissociation of the CheX2
dimer in the CheX-mediated dephosphorylation of CheY3 also
remains to be determined. One possibility is that CheX2dimer
dissociation could function to expose a surface on CheX which
allows interaction with an auxiliary regulatory protein, as seen
with CheC and the Rap family of response regulator phos-
3could selectively bind monomeric CheX, thus
Materials and Methods
Expression Plasmids, Protein Expression and Protein Purification. Plasmid deriv-
atives of pQE30 (Qiagen Inc.) encoding His-tagged CheX and CheY3 have
been described (20). Plasmids encoding CheX E96A and CheX N99A were
generated from the wild-type cheX plasmid by site directed mutagenesis
(QuikChange, Stratagene). Plasmids were transformed into M15/pREP4 cells
for overexpression as described (20). For incorporation of seleno-methionine
(Se-Met) into CheX for crystal growth, the M15/pREP4.pQE30 CheX strain was
grown in minimal media supplemented with 19 amino acids (all except
methionine). L-seleno-methionine (100 mg per liter) was added immediately
before overnight induction at 30 °C. His-tagged CheX and CheY3 were
purified from crude lysates by using Ni2þ-NTA affinity chromatography ac-
cording to manufacturer’s instructions (Qiagen Inc.) followed by gel filtration
chromatography (Superose 12, GE Biosciences) in buffer containing 50 mM
Tris, pH 7.5, 150 mM NaCl. Protein concentration was determined by
absorbance at 280 nm. Extinction coefficients (0.237 ðmg∕mLÞ−1cm−1for
CheX and 0.255 ðmg∕mLÞ−1cm−1for CheY3) were estimated by using the
Protparam utility (http://ca.expasy.org).
Crystallization and Data Collection. Crystals were prepared in a sitting drop
format by using Microbridges (Hampton Research). Drops contained 2 μL
of reservoir buffer (0.1 M sodium citrate, pH 5.6 and 1.5 M ammonium sul-
fate) and 2 μL of a mixture of CheY3 (2.6 mg∕mL), SeMet-CheX (2.5 mg∕mL),
20 mM MgCl2, 0.75 mM BeCl2, and 20 mM NaF. The protein mix also
contained Tris buffer (23 mM) and NaCl (69 mM) due to contribution from
protein stock solutions. Crystals required 1–2 weeks to grow to maximal size
(200 μm × 90 μm × 90 μm). Crystals were cryoprotected by using Fomblin Y
LVAC 14∕6 perfluoropolyether vacuum pump oil (Aldrich) and flash frozen
in liquid nitrogen. A single anomalous diffraction (SAD) dataset was collected
at the peak wavelength of Se (0.97923 Å) to a resolution of 1.96 Å at
the Advanced Photon Source at Argonne National Labs, Beamline 22-ID
Structural Determination and Refinement. The dataset was processed with the
HKL-2000 suite (31) and the structure was solved by using ShelXD (32) and
RESOLVE (33, 34). Four of the six Se-Met residues were located and initial
models were created manually by using E. coli CheY · BeF3· Mn2þ(pdb
1FQW) and T. maritima CheX (pdb 1XKO) based on the location of the Se
atoms. SWISS-MODEL homology modeling program (35) (http://swissmodel.
expasy.org/) was then used to create an updated model which contained the
B. burgdorferi CheY3 and CheX sequences. We incorporated the new model,
by using rigid body refinement in Refmac5 from CCP4 (36), to orient it in the
high resolution SAD maps and then initiated refinement. Manual model
building was performed by using Coot (37) and further restrained
refinement was carried out by using Refmac5 in CCP4 (36). The final stages
of model building and refinement were carried out by using O (38) and Crys-
tallography and NMR System (39), respectively. Crystallization parameters are
in Table S1.
Measurement of CheX Phosphatase Activity. Inorganic phosphate release was
measured in reactions containing CheY3, CheX, and the phosphodonor
monophosphoimidazole by using a spectroscopic enzyme-linked assay
(EnzChek Pi, Invitrogen), as previously described (25).
Molecular Weight Determination. His-tagged CheX was eluted off the Ni2þ-
NTA agarose column, dialyzed at 4°C versus 2 × 500 mL of buffer (20 mM
Hepes, pH 7.0, 200 mM NaCl, and 1 mM EDTA), concentrated to
∼2 mg∕mL, and filtered. The sample (100 μL) was then chromatographed
on a Superdex 200 10∕30 column (GE Healthcare) at 0.5 mL∕min in the same
buffer used for dialysis. The column eluent was fed directly into a DAWN EOS
multiangle light scattering spectrometer, followed by an OptiLab rEX Refrac-
tive Index detector, and a quasi-elastic light scattering module Wyatt QELS
(Wyatt Technology Corporation). The light scattering and refractive index
outputs were recorded continuously and the data combined to determine
the polydispersity and the number average molecular weight.
Genetic Complementation Experiments. Shuttle vectors expressing CheX E96A
and CheX N99A were constructed and transformed into B. burgdorferi cheX::
kan cells as described for wild-type CheX (20). Western blots demonstrated
that cells transformed with the mutant and wild-type cheX genes expressed
similar levels of CheX. Dark field microscopy was carried out as described
in ref. (20).
In the recent structure of a T. maritima histidine kinase/receiver
domain complex (40), the orientation of the receiver domain and
the kinase resembles that of the three previously solved receiver
domain-containing complexes discussed here.
ACKNOWLEDGMENTS. We are indebted to Robert Bourret for expert review of
the manuscript and Brenda Temple, Laurie Betts, and Shawn Williams for
advice with structural solution and analysis. We thank Ashutosh Tripathy
(UNC Macromolecular Interactions Facility) for assistance with light scattering
and the Advanced Photon Source at Argonne National Labs for X-ray data
collection. This work was supported by National Institutes of Health
Grants GM050860 (to Robert B. Bourret), AR054582 (to M. A. M.), AI29743
(to N. W. C) and GM080334 (to R. Z.).
www.pnas.org/cgi/doi/10.1073/pnas.0911185107Pazy et al.
1. Laub MT, Goulian M (2007) Specificity in two-component signal transduction path-
ways. Annu Rev Genet, 41:121–145.
2. Stock AM, Robinson VL, Goudreau PN (2000) Two-component signal transduction.
Annu Rev Biochem, 69:183–215.
3. Calva E, Oropeza R (2006) Two-componentsignal transduction systems, environmental
signals, and virulence. Microb Ecol, 51:166–176.
4. Bahn YS (2008) Master and commander in fungal pathogens: the two-component
system and the HOG signaling pathway. Eukaryot Cell, 7:2017–2036.
5. Gao R, Mack TR, Stock AM (2007) Bacterial response regulators: Versatile regulatory
strategies from common domains. Trends Biochem Sci, 32:225–234.
6. Gao R,StockAM(2009) Biologicalinsightsfromstructuresoftwo-componentproteins.
Annu Rev Microbiol 133–154.
7. West AH, Stock AM (2001) Histidine kinases and response regulator proteins in
two-component signaling systems. Trends Biochem Sci, 26:369–376.
8. Kirby JR, et al. (2001) CheC is related to the family of flagellar switch proteins and acts
independently from CheD to control chemotaxis in Bacillus subtilis. Mol Microbiol,
9. Zhao R, Collins EJ, Bourret RB, Silversmith RE (2002) Structure and catalytic mechanism
of the E coli chemotaxis phosphatase CheZ. Nat Struct Biol, 9:570–575.
10. Perego M, et al. (1994) Multiple protein-aspartate phosphatases provide a mechanism
for the integration of diverse signals in the control of development in B. subtilis. Cell,
11. Porter SL, Roberts MA, Manning CS, Armitage JP (2008) A bifunctional kinase-
phosphatase in bacterial chemotaxis. Proc Natl Acad Sci USA, 105:18531–18536.
12. Zhu Y, Qin L, Yoshida T, Inouye M (2000) Phosphatase activity of histidine kinase EnvZ
without kinase catalytic domain. Proc Natl Acad Sci USA, 97:7808–7813.
13. Ohlsen KL, Grimsley JK, Hoch JA (1994) Deactivation of the sporulation transcription
factor Spo0A by the Spo0E protein phosphatase. Proc Natl Acad Sci USA,
14. Muff TJ, Ordal GW (2008) The diverse CheC-type phosphatases: chemotaxis and
beyond. Mol Microbiol, 70:1054–1061.
15. Park SY, et al. (2004) Structure and function of an unusual family of protein phospha-
tases: the bacterial chemotaxis proteins CheC and CheX. Mol Cell, 16:563–574.
16. Szurmant H, Muff TJ, Ordal GW (2004) Bacillus subtilis CheC and FliYare members of a
novel class of CheY-P-hydrolyzing proteins in the chemotactic signal transduction
cascade. J Biol Chem, 279:21787–21792.
17. Muff TJ, Ordal GW (2007) The CheC phosphatase regulates chemotactic adaptation
through CheD. J Biol Chem, 282:34120–34128.
18. Muff TJ, Foster RM, Liu PJ, Ordal GW (2007) CheX in the three-phosphatase system of
bacterial chemotaxis. J Bacteriol, 189:7007–7013.
19. Charon NW, Goldstein SF (2002) Genetics of motility and chemotaxis of a fascinating
group of bacteria: the spirochetes. Annu Rev Genet, 36:47–73.
20. Motaleb MA, et al. (2005) CheX is a phosphorylated CheY phosphatase essential for
Borrelia burgdorferi chemotaxis. J Bacteriol, 187:7963–7969.
21. Ge Y, Charon NW (1997) Molecular characterization of a flagellar/chemotaxis operon
in the spirochete Borrelia burgdorferi. FEMS Microbiol Lett, 153:425–431.
22. Varughese KI, Tsigelny I, Zhao H (2006) The crystal structure of beryllofluoride
Spo0F in complex with the phosphotransferase Spo0B represents a phosphotransfer
pretransition state. J Bacteriol, 188:4970–4977.
23. Zhao X, Copeland DM, Soares AS, West AH (2008) Crystal structure of a complex
between the phosphorelay protein YPD1 and the response regulator domain of
SLN1 bound to a phosphoryl analog. J Mol Biol, 375:1141–1151.
24. Wang W, et al. (2002) Structural characterization of the reaction pathway in
phosphoserine phosphatase: crystallographic "snapshots" of intermediate states.
J Mol Biol, 319:421–431.
25. Silversmith RE, Levin MD, Schilling E, Bourret RB (2008) Kinetic characterization of
catalysis by the chemotaxis phosphatase CheZ. Modulation of activity by the
phosphorylated CheY substrate. J Biol Chem, 283:756–765.
26. Gherardini PF, Wass MN, Helmer-Citterich M, Sternberg MJ (2007) Convergent evolu-
tion of enzyme active sites is not a rare phenomenon. J Mol Biol, 372:817–845.
27. Doolittle RF (1994) Convergent evolution: The need to be explicit. Trends Biochem Sci,
28. Grenha R, et al. (2006) Structural characterization of Spo0E-like protein-aspartic acid
phosphatases that regulate sporulation in bacilli. J Biol Chem, 281:37993–38003.
29. Diaz AR, et al. (2008) Functional role for a conserved aspartate in the Spo0E signature
motif involved in the dephosphorylation of the Bacillus subtilis sporulation regulator
Spo0A. J Biol Chem, 283:2962–2972.
30. Chao X, et al. (2006) A receptor-modifying deamidase in complex with a signaling
phosphatase reveals reciprocal regulation. Cell, 124:561–571.
31. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collection in oscilla-
tion mode. Methods Enzymol, 276:307–326.
32. Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr A, 64:112–122.
33. Terwilliger TC (2000) Maximum-likelihood density modification. Acta Crystallogr D,
34. Terwilliger TC, Berendzen J (1999) Automated MAD and MIR structure solution. Acta
Crystallogr D, 55:849–861.
35. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: Aweb-
based environment for protein structure homology modelling. Bioinformatics,
36. Potterton E, Briggs P, Turkenburg M, Dodson E (2003) A graphical user interface to the
CCP4 program suite. Acta Crystallogr D, 59:1131–1137.
37. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta
Crystallogr D, 60:2126–2132.
38. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building
protein models in electron density maps and the location of errors in these models.
Acta Crystallogr A, 47:110–119.
39. Brunger AT, et al. (1998) Crystallography & NMR system: A new software suite for
macromolecular structure determination. Acta Crystallogr D, 54:905–921.
40. Casino P, Rubio V, Marina A (2009) Structural insight into partner specificity and
phosphoryl transfer in two-component signal transduction. Cell, 139():325–336.
Pazy et al.PNAS
February 2, 2010