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INTRODUCTION |
Escherichia coli motility involves short periods (1-2
s) of smooth swimming ("runs") punctuated by briefer episodes
(0.1-0.2 s) of somersaulting ("tumbles") that result in random
direction changes. As an E. coli cell swims about, its
chemotaxis system monitors the levels of attractant and repellent
chemicals encountered and modulates the turning probability to promote
cell migration up concentration gradients of attractants and down
gradients of repellents (1, 2). Chemotactic movement results from
communication between cell surface chemoreceptor proteins and
components of the flagellar motors. Two complementary processes,
excitation and adaptation, contribute to this control by modulating the
intracellular level of a "tumble signal," phosphorylated CheY
(3-6).
The molecular machinery that mediates excitation and adaptation in this
system has been defined and its mechanism of action elucidated in part:
transmembrane receptor proteins (also called MCPs)1 bind attractant and
repellent ligands in the periplasm and modulate the autokinase activity
of CheA (a protein histidine kinase) accordingly. Binding of repellent
stimuli to MCPs is thought to stimulate CheA autokinase activity,
whereas attractant stimuli inhibit this activity (7-12).
Autophosphorylated CheA can, in turn, donate its phosphoryl group to
CheY (13-15), a modification that directs CheY to bind to the switch
components of the flagellar motor and thereby promote tumbling (4).
Another possible fate for the phosphoryl group of P-CheA is transfer to
CheB (13, 16, 17); phosphorylation of CheB stimulates its
methylesterase activity and therefore promotes sensory adaptation by
adjusting the signaling properties of the MCPs via removal of
methylester groups from the side chains of specific glutamate residues
(16, 17).
The events that enable MCP-mediated control of CheA autokinase activity
are thought to occur within a ternary complex that includes MCPs, CheA,
and CheW (9, 11, 18-22). Understanding how this complex forms and how
it mediates CheA regulation are, therefore, central to defining the
molecular mechanisms of both excitation and adaptation in the
chemotaxis system. Although many components of the bacterial chemotaxis
system have been studied in considerable detail (for reviews, see Refs.
5, 6, 23, and 24), little is known about the specific biochemical
contributions of CheW. This 167-amino acid protein (7) does not appear
to have any enzymatic activity by itself; nor does it share sequence or
structural similarities with motifs that do have catalytic activities.
But CheW clearly plays a crucial role in the bacterial chemotaxis
system, as indicated by the complete inability of cheW mutants to respond to any chemotactic stimuli (25). We are attempting to define how CheW contributes to chemotaxis signal transduction.
CheW has four known activities in vitro: (i) binding to
CheA; (ii) binding to MCPs, such as Tar; (iii) promoting formation of
CheA·CheW·MCP ternary complexes; and (iv) enabling MCPs to stimulate and/or inhibit CheA autokinase activity (9, 10, 12, 18-22,
26, 27). Based on these observations, current models depict CheW as an
"adapter protein" that serves to tether CheA to the MCPs (18, 20,
28). While such a simple adapter role would account for the known
in vitro activities of CheW, it is important to emphasize
that the relationships among these in vitro activities and
their roles in vivo have not been clearly established. In
particular, the functional importance of CheW's binding activities has
not been clearly established by, for example, determining whether
disrupting the CheW 
CheA binding interaction affects formation of
the CheA·CheW·MCP ternary complex and/or activation of CheA within
this complex.
In previous work (29), we followed a traditional mutational approach in
an attempt to investigate the functional significance of CheW binding
interactions with CheA and MCPs: we identified mutations that disrupted
CheW's ability to support chemotaxis, and then we examined the effect
of these mutations on the ability of the protein to bind to CheA and
Tar. Each of the mutants we isolated following this approach was
defective in formation of an activated ternary complex and exhibited
defects in both binding interactions. While those results
suggested that CheW's binding activities are indeed important for its
role in chemotaxis signal transduction, they did not allow us to
examine the effects of eliminating just one binding interaction at a
time; nor did they allow us to identify what segments of CheW might be
involved specifically in interactions with CheA and which with MCPs. To
examine more rigorously the role(s) of CheW's binding activities and
to begin defining potential interaction interfaces with CheA and Tar,
we turned to the DITA approach of Inouye et al. (30) with
the goal of identifying CheW mutants lacking just one of the two
binding activities. Our results, in conjunction with the recently
solved three-dimensional structure of CheW (31), indicate clustering of
mutation sites that cause disruption of CheW CheA interactions and
therefore suggest the location of a possible CheA binding interface on
CheW. In addition, our results help to further elucidate the CheW
surface that appears to mediate contacts with MCPs, first defined by
the genetic suppressor studies of Liu and Parkinson (26).
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EXPERIMENTAL PROCEDURES |
Materials and Assays--
5-Fluoroorotic acid (5-FOA) was
purchased from Toronto Research Chemicals, Inc. Fluorescein 5-maleimide
was purchased Molecular Probes, Inc. (Eugene, OR). All other chemicals
were purchased from standard sources and were of reagent grade. The
following procedures were carried out using previously published
procedures: purification of CheA, CheW, and CheY (13, 18, 29, 32); isolation of Tar-containing inner membrane vesicles (18, 33); fluorescence-monitored assays of CheW binding to CheA and Tar (29);
coupled assays of CheA activity (11, 21, 34); and swarm assays (35,
36).
Strains and Plasmids--
Saccharomyces cerevisiae
strain YCJ4 and plasmids pLEX202PL, pAS-Snf1, and pLex-bicoid were
kindly provided by Jeremy Thorner (University of California, Berkeley).
Strains RP3098 (
(flhA-flhD) (37)) and D392
(cheW (38, 39)) are Escherichia coli K-12 derivatives kindly provided by J. S. Parkinson (University of Utah) and F. W. Dahlquist (University of Oregon), respectively. Strain D392 was used for swarm assays, whereas strain RP3098, lacking
all chemotaxis genes, was used for purification of most of the proteins
used in this study.
Plasmid pGAD:CheW encoded a CheW-Gal4AD fusion and was created using
vector pGAD424 (CLONTECH). Wild type
cheW was PCR-amplified from pCW (27) using primers that
generated EcoRI and SalI sites upstream and
downstream, respectively, of cheW. The resulting PCR
fragment was digested with EcoRI and SalI and
then ligated into corresponding sites of pGAD424. Plasmid pLex:cTar
encoding cTar-LexA fusion was created by ligating a fragment of
tar encoding amino acids 257-553 into EcoRI and
BamHI sites of plasmid pLex202PL. Plasmid pGBT9:CheA
encoding CheA-Gal4BD fusion was created by inserting full-length
cheA into vector pGBT9 (CLONTECH, Inc.) between SmaI and SalI sites.
The plasmid pCE (29) was used for overexpression and purification of
CheW mutant proteins carrying a Met-His6-Cys extension at
their N-terminal ends. Mutant cheW alleles were excised from corresponding pGAD:CheW plasmids and ligated into pCE using
EcoRI and SalI sites. For use in the swarm
assays, mutant cheW alleles were excised from pGAD:CheW
vectors using AgeI and SalI and ligated into
corresponding sites of pCnoW (29). All plasmids created in the course
of this work were verified by dideoxy sequencing (performed at the
University of Maryland Center for Agricultural Biotechnology).
Mutagenesis and Screening--
Random cheW mutants
were generated using an error-prone PCR procedure in which an unequal
ratio of dNTPs and a high concentration of Mn2+ created
conditions favorable for nucleotide misincorporation by Taq
polymerase (40). The PCR-generated, mutagenized cheW DNA
fragment was gel-purified and then transformed along with EcoRI/SalI-digested pGAD424 into YCJ4 strain
carrying pGBT9:CheA and pLex:cTar (30). pGAD:CheW plasmid was restored
in vivo in the resulting YCJ4 transformants by a
gap repair mechanism (41). Half of the transformation mixture was
plated on Yc medium lacking histidine, tryptophan, and leucine
(Yc
his,trp,leu) (selecting for pLex:cTar, pGBT9:CheA, and pGAD:CheW,
respectively) and including 5-FOA. Colonies capable of growth in
the presence of 5-FOA were transferred to Yc-his,trp,leu plates lacking
uracil, and the results of growth on these plates were evaluated after
3 days at 30 °C. The colonies that displayed a URA
phenotype were then restreaked on Yc-his,trp,leu plates; after a 2-day
incubation at 30 °C, these colonies were tested for
-galactosidase activity using a colony lift assay (42). The second
half of the transformation mixture was plated on
Yc-his,trp,leu medium in which the amount of uracil
was reduced from the normal level of 0.01 to 0.0008%; this selected
for URA+ colonies (attempts to use medium completely
lacking uracil were unsuccessful). Among the mutants growing on this
type of medium, we then identified -Gal colonies as
described above. pGAD:CheW from selected colonies was then isolated and
retransformed into YCJ4 strain expressing cTar and CheA fusions. The
resulting transformants were assayed for URA3 and
lacZ expression to confirm the phenotype of the strains observed in the differential interaction trap assay. Nucleotide changes
in these alleles were determined by dideoxy sequencing at the
University of Maryland.
Measuring CheW Affinities for CheA and Tar--
These assays
utilized wild-type and mutant CheW proteins that had been engineered to
include an N-terminal His6-Cys tag, as described previously
(29). The purified His6-Cys-CheW proteins were modified by
covalently attaching fluorescein maleimide (Molecular Probes) to the
single cysteine side chain. Fluorescently labeled His6-Cys-CheW (hereafter referred to as F*-CheW) was then
used for binding assays. Binding of wild-type or mutant F*-CheW to CheA
was monitored using fluorescence anisotropy as described previously
(29). In short, these assays involved measuring the increase in
fluorescence anisotropy (
ex = 492 nm, em = 517 nm) of the F*-CheW sample as increasing concentrations of CheA
were added. These experiments were performed in TEND buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 100 mM NaCl, 0.5 mM dithiothreitol) containing 10%
polyethylene glycol 12,000. For each CheW variant, the magnitude of the
observed anisotropy increase as a function of the CheA concentration
was used to generate a binding isotherm that was fitted to a simple
binding equation to generate an estimate of
KdCheA, the dissociation constant for
the F*-CheW·CheA complex. Details of this assay and analysis
procedure have been published previously (29).
Assays of the binding of wild-type or mutant CheWs to Tar were
performed using the "pull-down competition" approach detailed previously (29). These assays made use of inner membrane vesicles carrying overproduced levels of Tar (18). Such vesicles are easily
pelleted by centrifugation and carry with them, into the pellet,
F*-CheW in the expected amounts based on CheW binding to Tar. To assess
binding of mutant CheW proteins binding to Tar, competition experiments
were performed in which wild-type F*-CheW (final concentration 2 µM) was premixed with a particular CheW mutant protein
(0-50 µM). Then the inner membrane vesicles were added
to this mixture, generating a final Tar concentration of 11 µM in TEND buffer containing 1 mg ml1 BSA.
The resulting protein mixtures were allowed to equilibrate (in the
dark, with gentle mixing) for 10 min at 25 °C, and then the vesicles
(and associated F*-CheW) were sedimented by centrifugation. The
fluorescence emission intensity of the supernatants of these centrifuged samples were then measured (ex 492 nm,
em 517 nm). The data generated in these experiments
showed the expected progressive increase in signal intensity
(reflecting increasing levels of free F*-CheW) at increasing
concentrations of competitor (unlabeled, mutant CheW). Analyses of
these titration curves allowed us to define the Kd
of the complex formed by Tar with the mutant CheW (29).
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RESULTS |
Efficacy of the Differential Interaction Trap Assay--
We used
the DITA of Inouye et al. (30) to quickly and easily observe
CheW interactions with CheA and Tar. This approach is a three-hybrid
modification of the yeast two-hybrid assay (43), and it involved
simultaneous expression of three chimeric proteins, carried by three
distinct plasmids, in S. cerevisiae strain YCJ4. The three
chimeric fusions were as follows: CheW fused to the transcription
activation domain of Gal4 (CheW-Gal4AD); CheA fused to the DNA-binding
domain of Gal4 (CheA-Gal4BD); and the cytoplasmic domain of Tar (amino
acids 257-553) fused to the DNA-binding protein LexA (cTar-LexA). As
depicted in Fig. 1, interaction of
CheW-Gal4AD with CheA-Gal4BD is expected to result in expression of the
URA3 reporter gene in YCJ4, and interaction of CheW-Gal4AD
with cTar-LexA is expected to drive expression of the lacZ
reporter gene in this yeast strain. Our goal was to use this system to
identify two distinct classes of cheW mutations: (i) those
that diminished CheW binding to CheA, but not to Tar and (ii) those
that decreased the affinity of CheW for Tar but not for CheA. Before
proceeding with such a mutant search, we first examined the specificity
and sensitivity of the DITA two-hybrid interactions.
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Fig. 1.
Schematic diagram of the differential
interaction trap assay (30). Fusions of CheW to the Gal4
activation domain (Gal4AD), CheA to the Gal4 DNA-binding domain
(Gal4BD), and cTar (amino acids 257-553) to LexA (a DNA-binding
protein) were expressed in the yeast strain YCJ4. This strain has two
chromosomally integrated reporter genes: lacZ, located
downstream of the lexA operator, and URA3,
located downstream of the GAL4 UAS (30). Pairwise CheW Tar and CheW CheA interactions were assessed by expression of
lacZ and URA3, respectively.
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As expected, YCJ4 cells expressing CheW-Gal4AD, CheA-Gal4BD, and
cTar-LexA were Ura+ (5-FOA sensitive) and
-Gal+. The following controls were performed: YCJ4 cells
expressing just CheA-Gal4AD and cTar-LexA (but not CheW-Gal4AD) were
Ura, 5-FOA-resistant, and -Gal; YCJ4
cells expressing just CheW-Gal4AD and CheA-Gal4BD (but not cTar-LexA)
were Ura+, 5-FOA-sensitive, and -Gal; and
YCJ4 cells expressing just CheW-Gal4AD and cTar-LexA (but not
CheA-Gal4BD) were Ura, 5-FOA-resistant, and
-Gal+. We performed several additional control
experiments to assess the specificity of the apparent interactions of
CheW with CheA and Tar. In these, we detected no observable interaction
between CheW-Gal4AD and bicoid-LexA or Snf1-Gal4BD (30). These results indicated that the DITA can be used to accurately monitor CheW CheA
and CheW Tar binding interactions independently in the same cell.
To assess the sensitivity of the DITA in our system, we evaluated the
effects of six cheW mutations that we had previously shown
to cause moderate to severe decreases in the affinity of CheW for CheA
and Tar (29). Our results (Table I)
indicate that expression of the URA3 reporter gene is
sensitive to moderate (and large) changes of
KdCheA but not to small affinity
changes: a 6.5-fold increase in KdCheA
gave rise to a Ura phenotype, but a 3.3-fold increase did
not. We observed somewhat greater sensitivity for altered expression of
the lacZ reporter gene in response to changes in the
affinity of CheW for Tar; a 3-fold increase in
KdTar was sufficient to generate a
-Gal phenotype.
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Table I
Phenotypes of previously identified CheW mutant proteins in the
differential interaction trap assay and their affinities for CheA
and Tar
Affinities were determined using fluorescence anisotropy (to define
KdCheA) and competition pull-down assays
(to define KdTar) as detailed by
Boukhvalova et al. (29). Each value represents the average
of at least two independent titration experiments ± S.E.
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Tar and other MCPs form homodimers (44, 45) that appear to further
interact and form oligomers (e.g. trimers of dimers (28,
46)) that may represent the functional form of the chemoreceptors for
in vivo signaling. Cytoplasmic fragments of Tar (cTar) and Tsr (cTsr) also form such complexes (12, 47). However, it is not clear
whether MCP dimerization/oligomerization is necessary for CheW binding.
The dimerization status of the cTar-LexA fusion in our DITA experiments
is also unclear. LexA has the ability to form dimers. Although the
association constant for dimerization is relatively weak (2 × 104 M1) for LexA in isolation,
this affinity is improved dramatically upon binding to the
lexA operon (48, 49). Thus, LexA dimerization might promote
dimerization of our cTar-LexA fusion. Despite the potential
complexities associated with dimerization or higher order
oligomerization of cTar-LexA, our experiments indicated that the
two-hybrid interaction between cTar-LexA and CheW-Gal4AD served as an
accurate reporter of CheW Tar interaction: mutations in CheW that
affected in vitro binding to full-length Tar (in membrane vesicles) had the expected effect on the two-hybrid interaction.
Isolation of cheW Point Mutants Using DITA--
We subjected the
entire cheW gene to random mutagenesis using error-prone PCR
(40); PCR products were introduced into appropriately cleaved plasmid
pGAD424 (CLONTECH) and expressed as CheW-Gal4AD fusions in YCJ4 cells that already carried plasmids encoding
CheA-Gal4BD and cTar-LexA. To isolate variants of CheW that exhibited
weakened affinity for CheA, but not for Tar, we plated transformation
mixtures onto medium containing 5-FOA, a nucleotide analog that
inhibits growth of yeast cells expressing URA3 (50).
Colonies that grew on these plates were then tested for
-galactosidase activity. Of ~3,000 5-FOA-resistant colonies, nine
were found that maintained a -Gal+ phenotype (reflecting
maintenance of the CheW Tar interaction). These Ura
-Gal+ mutants (hereafter referred to as Class I) were
subjected to DNA sequence analysis and retained for further analysis.
To identify variants of CheW that exhibited diminished affinity for
Tar, but not for CheA, we first plated YCJ4 transformation mixtures
onto medium depleted of uracil and then screened ~2,000 of these to
identify four that were -Gal. These Ura+
-Gal mutants (hereafter referred to as Class II) were
subjected to DNA sequence analysis and retained for further analysis.
The nucleotide sequences of three of the Class II mutants exhibited
single point mutations, whereas the remaining Class II mutant and all
nine Class I mutants had two or more nucleotide substitutions. For each
of these multiple mutants, a single mutation was responsible for the
observed DITA phenotype. This was determined by separating the
mutations using convenient restriction sites and, in some instances, by
recreating individual mutations using oligonucleotide-directed
mutagenesis. The amino acid changes associated with the Class I and
Class II mutations are summarized in Table II.
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Table II
Amino acid changes in CheW mutant proteins isolated using the
differential interaction trap assay and affinities of mutants for
CheA and Tar
Affinities were determined using fluorescence anisotropy measurements
(to define KdCheA) and competition pull-down
assays (to define KdTar) as described
under "Experimental Procedures" and as detailed by Boukhvalova
et al. (29). Each value represents the average of at least
two independent titration experiments ± S.E.
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Biochemical Analysis of CheW Mutant Proteins--
The DITA results
suggested that specific mutations selectively inhibited CheW binding to
CheA or Tar. This approach, while extremely convenient for initial
identification of mutants, provided only an indirect and qualitative
assessment of binding defects for the CheW variants. To support these
results and quantify the effects of the mutations on CheW binding
affinities, we performed in vitro binding titrations that
utilized purified, fluorescein-labeled versions of the wild-type and
mutant variants of CheW (29). To evaluate CheW binding affinity for
CheA, we monitored the increase in fluorescence anisotropy exhibited by
labeled CheW when it binds CheA (Fig. 2)
(29). To assess CheW binding affinity for Tar, we used pull-down
experiments in which membrane vesicles carrying high levels of Tar were
used to sediment fluorescein-labeled CheW out of solution via
centrifugation (Fig. 3) (18, 29). These experiments defined binding isotherms that we analyzed to estimate KdCheA (the dissociation constant for
the CheW·CheA complex) and KdTar (the
dissociation constant for the CheW·Tar complex). The observed Kd values (Table II) indicated that the DITA had
successfully identified mutations that specifically diminished CheW
affinity for CheA (without affecting its affinity for Tar); Class I
mutants exhibited KdCheA values ranging
from 5- to 12-fold higher than the wild-type
KdCheA value, whereas
KdTar values for these mutants remained
within 30% of the wild-type value. Class II mutants, as expected,
exhibited significant increases in KdTar
(values ranged from 4 to 14 times the wild-type
KdTar value); however, this was
accompanied by a moderate (~3-fold) increase in
KdCheA. Several factors could have
contributed to this lack of specificity with the Class II mutants, such
as an inadequately sensitive reporter system for CheW CheA
interactions and/or the nature of the Tar binding interface, a
possibility that is considered in greater detail under
"Discussion."
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Fig. 2.
Fluorescence anisotropy analysis of CheW
binding to CheA. Increasing amounts of CheA were added to 0.8 µM solution of fluorescein-labeled wild type CheW ( )
or fluorescein-labeled CheW mutants E38D ( ), T46A ( ), or V45L
( ). Fluorescence anisotropy was measured after each CheA addition.
Results of titrations of F*-CheW with BSA ( ) are also shown. Binding
assays were carried out in TEND buffer, pH 7.5 (50 mM Tris,
0.5 mM EDTA, 0.1 M NaCl, and 0.5 mM
dithiothreitol), containing 10% polyethylene glycol 12,000. The
lines connecting the data points represent the best fits of
the data to a rectangular hyperbola defining the values of
KdCheA. This analysis and the details of
the titration procedures are as described in a previous paper
(29).
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Fig. 3.
Membrane vesicle pull-down assays of CheW
binding to Tar. A competition assay was used to monitor
displacement of fluorescein-labeled wild type CheW by mutant CheW
proteins. 2 µM F*-CheWwt was added to 11 µM Tar (in membrane vesicles) in the presence of
increasing amounts of unlabeled wild type CheW (), CheW mutants
L158Q (), V87A (), E38D (), or BSA () in TEND buffer in the
presence of 1 mg/ml BSA (added to minimize nonspecific association of
CheW with membrane vesicles). After a 10-min incubation at 25 °C,
membrane vesicles and bound proteins were sedimented by centrifugation,
and the fluorescence intensity remaining in the supernatant was
measured. Lines represent best fits of the data to define
the values of KdTar. Details of the
titration procedure and data analysis are described in a previous paper
(29).
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Effect of CheW Mutations on Tar·CheW·CheA Ternary
Complexes--
In the presence of CheW and MCPs, CheA exhibits a
dramatically enhanced autokinase activity (9, 11, 12, 21, 22, 51, 52).
This activation of CheA appears to result from formation of an
MCP·CheW·CheA ternary complex in which CheW serves as a "coupling
factor." Little is known about how CheW accomplishes this
receptor-kinase coupling or how it promotes formation of the ternary
complex. As a first step toward improving our understanding of the
functional role of CheW, we used our set of binding-defective CheW
proteins to determine whether both of CheW's binding interactions are
necessary for it to accomplish receptor-kinase coupling. We examined
the ability of our mutant CheW variants to accomplish CheA activation
by assaying CheA autokinase activity in mixtures containing purified
CheW, CheA, the cytoplasmic domain of Tar (LZ-cTar), and CheY in the
presence of an ATPase coupling system (34, 51, 52). These results (Fig.
4) indicate that each of our mutant CheW
variants was less effective than the wild-type CheW in mediating
MCP-CheA coupling in the concentration range tested. Although assays at
higher CheW concentrations would have been informative for some of the
mutant proteins, we were unable to pursue such assays because of
concentration limitations for CheW, CheA, and CheY; adding higher CheW
concentrations would have required corresponding decreases in the
volumes of CheA, CheY, and/or ATPase coupling reagents, but we were
unable to increase the stock concentrations of these proteins enough to
accommodate the necessary changes.
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Fig. 4.
Effect of CheW mutant proteins on CheA kinase
activity in the presence of Tar. Mixtures containing 11 µM CheA, 50 µM LZ-cTar (51, 52), 1 mg/ml
BSA, and 0-70 µM CheW were incubated at 30 °C for
3 h. Autokinase activity of CheA was then measured by an
enzyme-coupled ATPase assay (21, 34). A, CheA activation by
wild type CheW () and by CheW mutant proteins that are defective in
binding interactions with CheA: T46A (), V45L (), and K56I ().
B, CheA activation by wild type CheW () and CheW mutant
proteins that were initially identified as being defective in binding
interactions with Tar: V87A () and E38D (). A control reaction
carried in the absence of LZ-cTar () is shown in both
panels. Lines connecting the data points were
added to help the reader to distinguish among data sets and have no
theoretical significance.
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For the Class I mutants (Fig. 4A), we observed a consistent
qualitative correlation between the affinity of CheW for CheA and the
observed coupling effectiveness; mutant K56I had the highest KdCheA value and performed the worst in
the coupling assays; mutant T46A had the least affected
KdCheA value and was the most effective
Class I mutant in coupling assays. Other Class I mutants, with
intermediate KdCheA values, exhibited
intermediate coupling abilities. These results suggest that the binding
contacts that mediate formation of the CheW·CheA complex (in the
absence of MCP) are also involved in formation of the activated ternary complex.
Class II mutants also exhibited diminished coupling activities (Fig.
4B). However, attributing this decrease to a reduced affinity of CheW for Tar is complicated by the fact that these mutants
also have KdCheA values that are
~3-fold higher than the wild-type value. For assessing the effects of
an increase in KdTar, we found it
helpful to use the titration profile of mutant T46A (a Class I mutant)
as a standard for comparison; T46A exhibits a
KdCheA value that is ~6-fold higher
than the wild-type value and a KdTar
value that differs from the wild-type value by only ~30%. Both of
the Class II mutants that we studied in detail exhibited coupling abilities that were considerably worse than that of T46A. If the affinity of CheW for Tar had no effect on coupling ability, then we
would have expected Class II mutants to perform at least as well as
T46A and perhaps considerably better because they bound CheA somewhat
better than did T46A. This was not the case: Class II mutants E38D and
V87A both performed considerably worse than T46A in coupling assays.
These results suggest that the binding contacts that mediate formation
of the CheW·Tar complex (in the absence of CheA) are also involved in
formation of the activated ternary complex. We also noted that, in
contrast to the titration profiles observed with wild-type CheW and the
Class I mutants, the Class II mutants generated distinctly sigmoidal
titration profiles (Fig. 4B), perhaps reflecting
cooperativity, a possibility that is considered further under
"Discussion."
In Vivo Complementation by CheW Mutant Proteins with Altered
Binding Affinities--
To investigate how well our mutant CheW
proteins functioned in the context of the chemotaxis signaling pathway
in intact cells, we transformed plasmids expressing each
cheW allele into a cheW host strain and
assessed the chemotactic abilities of the resulting transformants by
examining their migration rates in semisolid "swarm" agar (Fig.
5). These results indicated that the
mutant variants were surprisingly effective, supporting swarm rates
that were close to that observed with wild-type CheW. These results indicate that in vivo activity of CheW is surprisingly
insensitive to moderate changes in the affinity of CheW for CheA and
MCPs.
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Fig. 5.
Complementation abilities of mutant CheW
variants. Swarm plate assays were carried out using E. coli strain D392 (cheW) expressing wild type CheW
(top gray line), no CheW
(bottom gray line), and the following
CheW mutant proteins: T46A (), V45L (), and K56I ()
(A); L158Q () (B); and V87A () or E38D
() (C). Transformants were inoculated into 0.3% tryptone
agar plates containing 0-50 µM
isopropyl--D-thiogalactoside (IPTG) and 100 µg ml1 ampicillin. The diameters of the outer edges of
swarm colonies were measured after 16 h of incubation at 30 °C.
Lines connecting the data points were added to help the reader to
distinguish among data sets and have no theoretical significance. The
reported swarm diameters are averages for at least two independent
experiments. Deviations in these values from one experiment to another
were small, such that the error bars for most
measurements were smaller than the size of the symbols used
in the figure. In a few cases, the error
bars were large enough to be shown.
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Under conditions of CheW overproduction, some differences were observed
with several of the mutants. Elevated levels of wild-type CheW resulted
in severe inhibition of chemotactic ability (Fig. 5), a phenomenon that
has been reported previously (14, 53). Class II mutants lacked this
inhibitory ability, whereas one of the Class I mutants (L158Q) appeared
to have enhanced inhibitory ability (Fig. 5B and data not
shown). L158Q exhibited an affinity for Tar that is slightly higher
(30%) than that of wild-type CheW. In previous work, we reported a
similar enhancement of inhibition with cheW154ocr, a mutant
that exhibited a 3-fold increased affinity for Tar compared with the
wild-type CheW (29). These results indicate a correlation between the
affinity of CheW for MCPs and its effectiveness as an inhibitor of
chemotaxis at elevated levels of expression; mutants with significantly
diminished affinity for Tar are ineffective as inhibitors, whereas
mutants with even moderately enhanced affinity for Tar are more
effective inhibitors.
| |
DISCUSSION |
Rationale, Advantages, and Limitations of the DITA Approach for
Analysis of CheW Binding--
To identify CheW mutants lacking just
one of the two binding activities, we applied the DITA approach of
Inouye et al. (30). One advantage of this
selection/screen is that it allowed us to focus specifically on
the protein-protein interactions of interest in a heterologous reporter
system without possible interference from other components of the
chemotaxis system or from unknown in vivo activities of
CheW. In addition, this approach selected against several types of
mutations that were uninteresting for our purposes, including those
that caused large scale perturbations of the three-dimensional
structure (e.g. mutants with disrupted folding patterns),
nonsense mutations, and mutations that caused significant changes in
CheW stability, turnover, or expression level.
This approach led to identification of several mutations (Class I) that
diminished CheW affinity for CheA without simultaneously affecting its
interaction with Tar. The existence of such mutants and their location
on the three-dimensional structure of CheW (see below) suggests that
the binding interfaces for CheA and Tar involve distinct faces of the
CheW surface. The DITA approach was less successful in identifying
mutations that specifically influenced CheW MCP binding
interactions; each of our Class II mutants exhibited a moderately
decreased ability to bind CheA in addition to the anticipated decrease
in affinity for Tar. Nonetheless, we were able to isolate one mutant
(E38D) that exhibited a large defect in Tar binding and a considerably
smaller defect in CheA binding. Our two classes of mutants allowed us
to address several basic questions about the functional role(s) of
CheW's binding interactions. These questions are considered below.
Mutations That Affect CheW Pairwise Binding Interactions Have
Corresponding Effects on Its Receptor-Kinase Coupling
Activity--
Titration of CheA with increasing concentrations of CheW
gives rise to a simple hyperbolic binding isotherm; there are no indications of multiple affinities of binding sites or cooperativity. Similar results are observed for titrations that monitor binding of
wild-type CheW to MCPs (18, 29, 52). We isolated mutants that exhibit
diminished binding affinities in these simple pairwise interactions,
and these mutants allowed us to address a simple question: Do
these mutations also affect the ability of CheW to promote
formation of the CheA·CheW·MCP ternary complex that is thought to
mediate key events in chemotaxis signal transduction? At first glance,
this question might seem trivial and its answer obvious. However, it is
important to emphasize the potential complexities of CheW binding
interactions in CheA·CheW·MCP complexes. In contrast to the
apparent simplicity of CheW pairwise binding interactions, when CheW is
placed in mixtures of CheA and MCPs, there appear to be multiple
competing binding equilibria that result in formation of several
distinct ternary complexes. These different versions of "the ternary
complex" have different stoichiometries, different kinase activities,
and different stabilities (21). This complexity suggests that the
component proteins can interact with one another in a variety of
different ways, some of which lead to a ternary complex in which CheA
autokinase activity is very high (the "activated ternary complex")
and some of which lead to a ternary complex in which CheA is
essentially inactive (the "inactive ternary complex"). In view of
this complexity, we deemed it important to investigate the relationship
between the pairwise interactions and those that mediate formation of
the activated ternary complex.
Our results clearly indicate that mutations that diminish the affinity
of the pairwise interactions have a corresponding effect on the ability
of CheW to promote formation of the activated ternary complex. In
theory, the differences observed between the wild-type and mutant
titration profiles (Fig. 4) could reflect either diminished levels of
activated ternary complex or diminished activity of CheA within this
complex. We did not pursue experiments to distinguish between these two
possibilities; however, it is notable that the shapes of the profiles
and the observed magnitudes of the maximal activities match fairly
closely with those predicted by computer simulations generated by
assuming diminished complex
formation.2 These results
suggest that CheW coupling activity involves both of its binding
interactions (i.e. CheW must interact with both CheA and Tar
to enable formation of the active complex). Moreover, these findings
are consistent with the idea that the binding contacts that mediate
pairwise CheW CheA and CheW Tar interactions are closely
related to (perhaps even identical to) the contacts that are required
for formation of the activated ternary complex.
Two of our mutant CheW proteins (V87A and E38D) generated a sigmoidal
relationship between the concentration of CheW and the levels of
activated complex, suggesting that activated complex formation is a
cooperative process, at least with these mutants (see Fig.
4B). We noted a similar effect with the mutant CheW154ocr in
previous work (29). CheW154ocr and the Class II mutants analyzed here
had altered affinity for receptors. However, pairwise titration experiments using these mutants and Tar gave rise to simple hyperbolic binding curves; only in the presence of CheA (in the kinase activation assays) did we observe the apparent cooperativity. Cooperativity in
regulation of CheA activity by receptors has been reported previously
(12, 21, 22, 54) and may be linked to the phenomenon of receptor
clustering (19, 45). Our results suggest that CheW might contribute to
this phenomenon through its binding interactions with receptors.
Binding of CheW to an MCP dimer might, for example, generate or uncover
additional contact sites that promote formation of a complex involving
CheW, CheA, and a cluster of MCPs. These "revealed sites" might
include MCP positions that become available for interaction with CheA,
similar sites on CheA that become available for interaction with MCPs,
or possibly some additional sites on CheW itself that allow it to act
as a nucleator of higher-order macromolecular aggregates. It is
puzzling that we observed apparent cooperativity with several CheW
mutant variants but not with other mutants and not with wild-type CheW.
Further detailed analysis of these mutants may provide insight into the
molecular mechanism by which CheW promotes formation of activated
ternary complexes.
In Vivo Chemotactic Signaling Tolerates Significant Decreases in
CheW Binding Affinities--
We examined the ability of our
binding-defective CheW variants to support chemotaxis in
vivo. When examining the results of these complementation
experiments, it is useful to consider two mutants as representative
test cases: K56I for examining the effect of diminishing the affinity
of CheW for CheA and E38D for examining the effect of diminishing the
affinity of CheW for Tar. The K56I mutant exhibits an affinity for CheA
that is weaker than that of wild-type CheW by a factor of ~12, but
its affinity for Tar is essentially the same as wild type. The E38D
mutant has a diminished affinity for Tar by a factor of 14, whereas its
ability to bind CheA is influenced to a lesser extent (~3-fold). Both
of these mutants exhibited diminished abilities to promote formation of the activated ternary complex in vitro, as discussed above.
Therefore, we were surprised to find that these and other such mutants
performed quite well when tested by in vivo complementation
(swarm) assays; the mutant proteins supported swarming rates that were
comparable with that supported by wild-type CheW. One conceivable,
albeit extreme, interpretation of these findings would be that in
vitro binding, ternary complex formation, and CheW coupling
activity are in vitro artifacts that have little to do with
CheW activity in vivo. However, in previous work we
demonstrated that several cheW null alleles (isolated by
virtue of their Che phenotype in swarm assays) encode
variants of the CheW protein that have severely disrupted binding
activities and that completely lack coupling activity in
vitro. This observation and the dramatic increase in CheA activity
that results from ternary complex formation (9, 21) argue against the
extreme interpretation suggested above. A second possibility is that
the chemotaxis signaling pathway is able to make due with a severely
diminished level of activated ternary complex. Several recent
publications have demonstrated that the signaling circuitry of the
chemotaxis system is a "robust" network that can maintain
responsiveness and sensitivity despite large fluctuations in the
activities and/or concentrations of key signaling components (55-57).
In previous work (58), we observed that CheA active site mutants having
autokinase activities as low as 6% of the wild-type activity can
support chemotaxis (as measured by swarm assays). Thus, the 10- or
20-fold reduction in the amount of activated ternary complex expected
for our E38D and K56I mutants might be accommodated by this robust
network. Such a situation would explain why most cheW null
alleles (isolated by screening for a Che phenotype (29))
have very large effects on both KdCheA
and KdTar; less drastic effects on CheW
binding would have no phenotype in swarm assays and would never be
identified for further analysis. Yet a third possible explanation of
the surprising efficacy of our weakly binding CheW mutants is that
in vivo conditions compensate in some other way for the poor
binding abilities of the mutant CheW proteins, perhaps as a result of
macromolecular crowding in the cytoplasm (59). Receptor clustering (19,
60) or adjustments of MCP methylation levels (55, 61, 62) might also
contribute to the ability of the in vivo chemotaxis system
to overcome defects in CheW binding affinity. However, our qualitative
examination of MCP methylation patterns in cells expressing these
cheW alleles did not reveal any clear differences from
methylation patterns observed in cells expressing wild-type
cheW, leading us to doubt the contribution of methylation as
a compensating factor (results not shown).
Mutation Sites Define Possible Interaction Interfaces for CheW
Binding to CheA and MCPs--
One of the goals of this study was to
define the segments of CheW that mediate its binding to CheA and to
MCPs. The results discussed above suggest that the binding surfaces
used in pairwise CheW·CheA and CheW·Tar complexes are also utilized
in the activated ternary complex, so identifying sites that are
required for pairwise binding interactions is a viable approach for
defining sites that play important roles in the protein-protein
interactions of the activated ternary complex. The three-dimensional
structure of CheW from Thermotoga maritima has recently been
determined by NMR methods in the Dahlquist laboratory (31). This
structure indicates that CheW consists of two five-stranded -barrels
surrounding a hydrophobic core (Fig. 6).
The T. maritima protein has an amino acid sequence that is
quite similar to that of the E. coli protein (63);
therefore, it seems likely that they have similar structures. Such
similarity has been observed in the structures of another component of
the chemotaxis system (CheY) from E. coli and T. maritima (64). Mapping our mutation sites onto the structure of
T. maritima CheW allowed us to visualize the relative
orientation, in three-dimensional space, of the amino acid positions
altered by our Class I and Class II mutations. The most striking result from such analysis is the clustering of the Class I mutation sites on
one face of CheW (Fig. 6A). In particular, these sites
cluster along the solvent-exposed edges of -strands 4 and 5, an
extended loop that connects 3 to 4, and
the C-terminal 
-helix. We propose that this surface of CheW serves
as a binding interface that mediates interactions between CheW and CheA
in pairwise combinations as well as in the activated ternary complex.
The same surface of CheW was proposed to mediate its interaction with
CheA based on NMR chemical shift changes observed in T. maritima CheW in the presence of CheA (31). Possible CheA CheW
interaction sites have also been proposed as a result of computer
models generated by assuming that CheW adopts a three-dimensional
structure resembling that of the C-terminal regulatory domain of CheA
(28, 65). Our results support the prediction of Bilwes et
al. (65) that the 3-4 region of CheW
(corresponding to 10 and 11 of CheA) mediates CheW CheA binding interactions.
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Fig. 6.
Proposed CheW CheA and CheW Tar
interaction interfaces on NMR-derived structure of T. maritima CheW (31). A, CheA interaction
interface of CheW defined by our Class I mutants. Amino acid residues
identified as important for CheW CheA interaction are
depicted in space-filling mode using red. B, Tar
interaction interface of CheW. Amino acid residues important for CheW
Tar interaction identified by our Class II mutants are shown in
green. Positions identified by Liu and Parkinson (26)
through genetic suppression studies are shown in yellow.
This figure was prepared using WebLab ViewerPro
4.0 and the averaged structural coordinates from 20 NMR-derived
structures of T. maritima CheW kindly provided by Griswold
and Dahlquist (University of Oregon) (31) and now available via the
Protein Data Bank (1KOS).
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We were successful in identifying only four Class II mutants. With such
a small sample size, "clustering" of these on the three-dimensional
structure of CheW (Fig. 6B) is not necessarily a surprising
or insightful finding. However, there are numerous cheW mutations that were identified by Liu and Parkinson
(26) as suppressors of MCP mutations. Presumably, these mutations
affect positions that participate in CheW-MCP contacts. Mapping these mutation sites and our Class II sites onto the structure of T. maritima CheW defines an extended surface adjacent to the putative CheA binding site of CheW (Fig. 6). We suggest that this extended surface mediates CheW MCP binding in both the pairwise CheW MCP
complexes as well as in the activated ternary complex.
Our approach identified mutations that altered CheW CheA or CheW
Tar binding. Several different types of changes in protein structure might underlie the observed binding defects: removal of
binding contacts, steric disruption of binding contacts, nonspecific changes in global structure, and/or protein folding. In this regard, it
is important to note that the Class I mutants exhibited normal affinities for Tar, so it seems unlikely that the binding phenotype of
these mutants (diminished affinity for CheA) arose from nonspecific alterations of CheW structure. The results generated with the Class II
mutants are less clear cut; these mutations had the expected effects on
Tar binding affinity but also caused small decreases in the affinity of
CheW for CheA. The Class II mutations affect -strands located on a
common surface of CheW and might cause subtle structural changes in the
orientations of these strands, changes that could be propagated to the
CheA-binding interface of CheW. In this regard, it is interesting to
note that the C-terminal -helix of CheW and -strand 10 (connected
to the N-terminal end of this helix) together form a structure that
runs along the entire length of the CheW molecule and contacts most of
the other secondary structural elements. This might provide a physical
link that conveys structural perturbations (such as those caused by
mutations) from the MCP-binding interface of CheW to its CheA-binding
interface. Deleting most of this helix (in CheW154ocr) has the
interesting effect of enhancing the affinity of CheW for Tar by a
factor of 3 (compared with wild-type CheW) (29). Perhaps, in wild-type CheW, this helix mediates communication between the two binding interfaces of the protein.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 301-405-5475;
Fax: 301-314-9489; E-mail: rs224@umail.umd.edu.
