(Received for publication, November 10, 1995; and in revised form, January 16, 1996)
From the
The phosphorylated form of the response regulator CheY promotes the tumble signal in Escherichia coli chemotaxis. Phospho-CheY is thought to interact with the switch at the base of the flagellar motor and cause reversal of flagellar rotation from counterclockwise to clockwise changing the swimming direction. Thus the level of phospho-CheY controls the direction of flagellar rotation. The decay of the tumble signal is caused by dephosphorylation of CheY. CheY has an intrinsic autophosphatase activity; however, this reaction is greatly accelerated by the presence of the CheZ protein.
We have shown previously that mutations at residues Asn-23 and Lys-26 in CheY confer resistance to the dephosphorylation activity of CheZ (Sanna, M. G., Swanson, R. V., Bourret, R. B., and Simon, M. I.(1995) Mol. Microbiol. 15, 1069-1079). Here we show that mutant CheY(N23D) is impaired in binding to CheZ, which provides a possible explanation for its resistance to the dephosphorylation activity of CheZ. Moreover, we isolated CheZ second-site suppressors of CheY(N23D), which restore both dephosphorylation and binding activity in a CheY(N23D) background. When the CheZ suppressor mutations are mapped, they are found in two clusters at the N and C termini of the CheZ protein which could define two regions of interaction with CheY. Furthermore, these regions may generate a surface in the folded three-dimensional structure of CheZ required for interaction with CheY.
The ability of Escherichia coli to survive in different environments may be the result of its ability to swim toward favorable conditions using the rotation of 6-8 flagella. This process is called bacterial chemotaxis and has been extensively studied in the past 30 years. When flagella rotate counterclockwise, they promote swimming in one direction for an extensive period of time (smooth swimming); reverse rotation (clockwise) causes tumbling of the cell, thus reorienting the swimming direction. The signal transduction pathway that regulates bacterial chemotaxis consists of the transmembrane receptor proteins Tar, Tsr, Trg, and Tap; the two-component system CheA/CheY; and the CheW and CheZ proteins (for reviews see (1, 2, 3, 4) ). The external changes detected by the receptor are ``communicated'' to the histidine kinase CheA, which is able to modulate its autophosphorylation capacity(5, 6) . Phosphorylated CheA transfers the phosphate group to the response regulator CheY(7, 8) , which in the phosphorylated form is able to interact with the flagellar switch and modify the sense of flagellar rotation from counterclockwise to clockwise, promoting tumble motion(9, 10, 11, 12, 13) . The CheZ protein accelerates the dephosphorylation of CheY, thus restoring smooth swimming(7, 14, 15) .
The mechanism of interaction between CheY and CheZ has not been elucidated, although some observations suggest an allosteric effect of CheZ on CheY instead of canonical phosphatase activity(16) . We isolated a mutant CheY(N23D) that is resistant to the dephosphorylating activity of CheZ but is not impaired in phosphorylation or autodephosphorylation(16) , which suggests that the mutant CheY may have a wild-type conformation but could be impaired in its ability to interact with CheZ.
We now report direct evidence that the CheY(N23D) mutant has lower binding affinity for CheZ compared to the wild-type CheY protein. Also, in order to identify possible regions of CheZ that interact with CheY, we used the background CheY(N23D) to isolate CheZ suppressor mutants with restored dephosphorylation activity. The CheZ suppressor mutations we have isolated cluster in two regions that may define binding sites for the CheY protein.
pREP4 (Qiagen) carrying the wild-type or N23D cheY gene was obtained subcloning the cheY genes from plasmids pRBB40 and pRBB40.N23D(16) , respectively.
Single time point assays and kinetics of dephosphorylation of CheY were performed as described previously(16) .
In order to compare the affinity of mutant
CheY(N23D) and wild-type CheY for CheZ, His-tagged CheY and His-tagged
CheY(N23D) were immobilized on Ni-NTA beads and CheZ
was added in the presence or absence of acetyl phosphate
(AcPO
), which is a small phosphodonor molecule able to
phosphorylate CheY in the absence of CheA(20) . The amount of
CheZ bound to wild-type CheY and CheY(N23D) was detected by Western
blot using polyclonal antibody against CheZ. In the presence of
AcPO
, the amount of CheZ bound by CheY(N23D) is about
7-fold lower compared to the amount bound by wild-type CheY as
quantified by densitometry (Fig. 1); since we found that CheY
and CheY(N23D) are equally well phosphorylated, this suggests an
impaired capacity of phosphorylated CheY(N23D) to interact with CheZ.
It is also evident in this experiment that the amount of CheZ bound to
CheY is much higher in the presence of AcPO
compared to the
reactions in which the phosphate donor is omitted. This result suggests
that CheZ has a higher affinity to the phosphorylated form of CheY
compared to the unphosphorylated form and is consistent with recently
published results by Blat and Eisenbach(21) .
Figure 1:
Binding of CheZ to wild-type CheY and
CheY(N23D). Wild-type CheY or CheY(N23D) were incubated in the presence
(+) or absence(-) of phosphorylating agent
(AcPO). Then wild-type CheZ was added to the reaction. The
amount of CheZ bound to CheY or CheY(N23D) was detected by Western
blot. CheY was detected with Coomassie and Ponceau Red
staining.
Approximately 300 plates were screened by miniswarm assay looking for bacteria with restored swarming capacity (Fig. 2, A andB). The negative control strain MGS143, which carries cheYN23D and wild-type cheZ genes, is impaired in chemotaxis (Fig. 2, A, C (right), and D (top)), because it constantly tumbles, thus forming small colonies when assayed for swarm capacity on motility plates.
Figure 2:
Screening for cheZ suppressors of cheYN23D. A, miniswarm assay of control strain MGS143
(cheY/pREP4cheYN23D/PQE12cheZ wild-type), which is non-chemotactic (unable to swarm) and has
tumble (clockwise) bias swimming behavior. B miniswarm assay
of
cheY/pREP4cheYN23D/PQE12cheZ mutant
library; swarming colonies of cheZ suppressor mutants were
isolated (examples are indicated by the arrows). C, swarm
assay of control strains; top, RP437recA (wild-type
for chemotaxis); bottom, KO641recA (
cheY strain; this strain is non-chemotactic with smooth
(counterclockwise) bias swimming behavior); left, MGS141
(
cheY/pREP4cheYwild-type/pQE12cheZ wild-type; this strain is partially chemotactic with slightly
smooth bias swimming behavior); right, MGS143
(
cheY/pREP4cheYN23D/pQE12cheZ wild-type; this strain is dramatically impaired for chemotaxis
with strong tumble bias swimming behavior). D, top,
MGS143 (as in C (right)); other panels,
MGS138/pQE12cheZ mutant
suppressors.
Single colonies were isolated from 440 selected swarming strains, assayed on motility agar, and tethered on glass coverslips to confirm their phenotype. All the potential suppressor strains were found to be better swarmers than the negative control (Fig. 2D; control swarming strains are shown in Fig. 2C), and they showed either wild-type or counterclockwise bias of flagellar rotation (data not shown). Among the 440 selected clones, 20 strains were chosen for further analysis because they showed different capacities for restoring chemotaxis.
Figure 3:
Distribution of cheZ mutations
isolated as suppressors of cheYN23D mutation. The amino acid
substitutions encoded by cheZ suppressor mutations are
clustered primarily in two regions of the CheZ protein. Mutations are
named with the residue number followed by the single-letter
abbreviation for the wild-type amino acid and the mutant amino
acid. The length of the vertical lines is proportional to the in vivo suppression capacity of each mutant, which was
calculated as their capacity to restore swarming in the cheYN23D background (i.e. L24P strong suppression
activity, diameter of the swarming colony 2.7 cm; R29C weak
suppression activity diameter of the swarming colony
1.0 cm;
diameter of the MGS143 swarming colony
0.7 cm). The suppressors
chosen for in vitro analysis are boxed.
Interestingly, the suppressor mutations are distributed on CheZ in two regions that seem to define separate clusters (Fig. 3). We selected five mutants for in vitro analysis, considering that they were candidates from both regions and that they showed different degrees of suppression in vivo. The in vivo suppression capacity of the CheZ mutants was quantified by measuring the diameter of the swarming colonies (Fig. 3).
Figure 4:
Dephosphorylation of phospho-CheY(N23D) by
CheZ suppressors. A, image of phosphorylated CheY(N23D) after
incubation with 4 pmol of purified wild-type or suppressor CheZ
proteins. Phosphorylated CheY(N23D) in the absence of CheZ is also
shown for comparison as well as wild-type phospho-CheY with or without
wild-type CheZ. B, graphic representation of different amounts
of phosphorylated wild-type or CheY(N23D) as quantified with a
PhosphorImager (Molecular Dynamics). Amounts are expressed as
PhosphorImager units and represent the mean of three separate
experiments. The standard deviation was <8%. C, cold chase
dephosphorylation kinetics of phospho-CheN23D mediated by CheZ mutant
suppressors. 0.5 pmol of wild-type or mutant CheZ were used in the
assays. , no CheZ; &cjs2134;, wild-type CheZ;
, CheZ(V166E);
, CheZ(R29C);
, CheZ(N170D);
, CheZ(L28P); ,
CheZ(L24P).
Based on cold-chase experiments the estimated tfor phosphorylated CheY(N23D) in the presence of
CheZ(L24P) and CheZ(L28P) is
10 and
40 s for CheZ(V166E),
CheZ(N170D), and CheZ(R29C) compared to
100 s for wild-type CheZ (Fig. 4C). The results of in vitro experiments
also parallel the results obtained in vivo; for example
CheZ(L24P) and CheZ(L28P) were found to be among the strongest
suppressors in vivo (see Fig. 3), and these proteins
exhibit the highest dephosphorylation activities on phospho-CheY(N23D) in vitro.
When the suppressors were assayed on wild-type phospho-CheY, four of them showed enhanced dephosphorylation capacity compared to wild-type CheZ (Table 1). Similar results were found when suppressors were incubated with mutant CheY(K26E), which was previously found to be partially resistant to the dephosphorylation of wild-type CheZ ( (16) and Table 1). Only mutant R29C showed weaker activity on wild-type CheY as well on CheY(K26E) when compared to wild-type CheZ.
Figure 5:
Binding of CheZ mutant suppressors to
CheY(N23D). The binding experiments were performed as described under
``Materials and Methods.'' All the reactions were done in the
presence of AcPO. Bands representing wild-type CheZ bound
to wild-type CheY or to CheY(N23D) are also shown for comparison (first two left lanes).
The CheY(N23D) mutant shows a specific lack of interaction
with CheZ. It is resistant to the dephosphorylating activity of CheZ
without being impaired in its phosphorylation or autodephosphorylation
activity, suggesting that a highly localized change in structure is
responsible for loss of activity. This is consistent with
two-dimensional NMR analysis in which the CheY(N23D) mutant does not
appear to have an altered overall conformation compared to the
wild-type CheY protein. ()We have found that the CheY(N23D)
mutant is impaired in CheZ binding. In order to study the contribution
of CheZ to the CheY-CheZ interaction, we have isolated CheZ suppressors
of the CheY(N23D) protein. The suppressors restore chemotactic behavior in vivo as well as in vitro dephosphorylation and
binding activity between CheZ and CheY(N23D).
Interestingly, two of the isolated CheZ suppressors of CheY(N23D) carried amino acid substitutions of residues 54R and 166V. Changes at these residues, CheZ(R54C) and CheZ(V166G), were previously reported by Huang and Stewart (22) as Salmonella typhimurium CheZ mutants with enhanced dephosphorylation activity on E. coli CheY. Although the identity of the specific amino acid changes were different, we also isolated mutants at residues 54 and 166 as suppressors of CheY(N23D) (Fig. 3). These mutants showed suppression activity in vivo, and mutant CheZ(V166E) was found to dephosphorylate the E. coli wild-type CheY at a rate slightly higher than that of wild-type CheZ (Table 1).
The assayed CheZ mutants showed different degrees of dephosphorylation of wild-type CheY ranging from 0.7 to 4.6 times the wild-type CheZ dephosphorylation rate. In particular, CheZ mutants V166E, N170D, L28P, and L24P were found to dephosphorylate wild-type CheY and CheY(K26E) better than wild-type CheZ, whereas CheZ(R29C) was partially impaired in its dephosphorylation activity (Table 1). These observations suggest that mutants V166E, N170D, L28P, and L24P are also gain-of-function CheZ mutants. Moreover these results suggest that suppressors V166E, N170D, L28P, and L24P are not allele specific for the N23D mutation, whereas R29C, which dephosphorylates CheY(N23D) better than wild-type CheZ but is less active on wild-type CheY or CheY(K26E), seems to show specificity for the N23D mutation.
There are at least three possible models to explain the suppression of the CheY(N23D) tumbly phenotype by the CheZ mutants. (i) the mutations are located on the CheZ protein in regions that are directly involved in the interaction with CheY; (ii) the mutations change the conformation of CheZ enhancing its binding to CheY; (iii) the oligomerization capacity is enhanced in the CheZ mutant suppressors. Each of the three proposed mechanisms would alter the rate of dephosphorylation and/or the substrate specificity. It has been recently shown (23) that CheZ forms oligomers in the presence of phosphorylated CheY. This property seems to enhance the dephosphorylating activity of CheZ, and it could represent a mechanism of CheZ regulation.
The suppressor
mutations map approximately on the CheZ protein in two regions rich in
leucine and isoleucine residues. It is known that leucine-rich
sequences can be involved in protein-protein interaction. The results
of a secondary structure prediction indicate that the suppressor sites
may be clustered in two -helices (Fig. 6). On the
hypothetical
-helices, the mutated residues would be located on
one face of each helix and overlapping with the leucine-rich and
hydrophobic region, which could be expected to be involved in the
interaction with other protein(s) (Fig. 6). If this is the case,
these regions of the
-helices may represent CheZ binding sites for
CheY, or form a continuous surface of interaction with CheY in the
folded three-dimensional structure of CheZ; an alternate hypothesis is
that they could be CheZ oligomerization sites.
Figure 6:
Distribution of mutated residues on
hypothetical -helical CheZ structures. The CheZ regions defined by
the suppressors mutations were predicted to be
-helical secondary
structures by the Chou-Fasman and Garnier-Robson algorithms (GCG
package). The Helicalwheel plot shows how the mutated residues (arrows) would be distributed defining a face on the
hypothetical
-helix 1 from Ile-17 to Ala-40 (left), which
represent the cluster of mutations at the N terminus of the CheZ
protein or
-helix 2 from Thr-145 to Asn-170 (right,
mutants at the C terminus) defining a potential surface of interaction
with CheY. Hydrophobic residues are boxed.