Phosphorylation of the response regulator CheV is required for adaptation to attractants during Bacillus subtilis chemotaxis.

In the Gram-positive soil bacterium Bacillus subtilis, the chemoreceptors are coupled to the central two-component kinase CheA via two proteins, CheW and CheV. CheV is a two-domain protein with an N-terminal CheW-like domain and a C-terminal two-component receiver domain. In this study, we show that CheV is phosphorylated in vitro on a conserved aspartate in the presence of phosphorylated CheA (CheA-P). This reaction is slower compared with the phospho-transfer reaction between CheA-P and one other response regulator of the system, CheB. CheV-P is also highly stable in comparison with CheB-P. Both of these properties are more pronounced in the full-length protein compared with a truncated form composed only of the receiver domain, that is, deletion of the CheW-like domain results in increase in the rate of the phospho-transfer reaction and decrease in stability of the phosphorylated protein. Phosphorylation of CheV is required for adaptation to the addition of the chemoattractant asparagine. In tethered-cell assays, strains expressing an unphosphorylatable point mutant of cheV or a truncated mutant lacking the entire receiver domain are severely impaired in adaptation to the addition of asparagine. Both of these strains, however, show near normal counterclockwise biases, suggesting that in the absence of the attractant the chemoreceptors are efficiently coupled to CheA kinase by the mutant CheV proteins. Inability of the CheW-like domain of CheV to support complete adaptation to the addition of asparagine also suggests that unlike CheW, this domain by itself may lead to the formation of signaling complexes that stay overactive in the presence of the attractant. A possible structural basis for this feature is discussed.     

Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex

Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a “phosphate sink” possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.     

Receptor conformational changes enhance methylesterase activity during chemotaxis by Bacillus subtilis

Addition and removal of the attractant asparagine causes methanol formation as a consequence of methylation and demethylation of conserved glutamate residues in the Bacillus subtilis chemotaxis receptor McpB C-terminal domain. We found that methanol was released on both addition and removal of asparagine even when the response regulator domain of CheB was removed (to produce CheB(141-357)). Thus, in undergoing the transition from unbound receptor to ligand-bound adapted receptor, the receptor must pass through a state of heightened susceptibility to demethylation by CheB that is independent of phosphorylation. The same result occurred when the aspartate phosphorylation site of CheB, Asp54, had been mutated to an asparagine residue, provided the enzyme was sufficiently induced. However, no methanol release was observed for an active site point mutant, cheB(S173C), in response to addition or removal of asparagine even when induced. Finally, methanol release was observed only for attractant addition in a mutant background lacking the coupling proteins, CheW and CheV, provided CheB(141-357) was present. Thus, on attractant addition, methanol must arise from a transient conformation of the receptor C-terminal domain that is an intrinsic property of the receptor; on attractant removal, however, methanol must arise from a different transient conformation, one dependent on the presence of coupling proteins.     

Attractant binding induces distinct structural changes to the polar and lateral signaling clusters in Bacillus subtilis chemotaxis

Bacteria employ a modified two-component system for chemotaxis, where the receptors form ternary complexes with CheA histidine kinases and CheW adaptor proteins. These complexes are arranged in semi-ordered arrays clustered predominantly at the cell poles. The prevailing models assume that these arrays are static and reorganize only locally in response to attractant binding. Recent studies have shown, however, that these structures may in fact be much more fluid. We investigated the localization of the chemotaxis signaling arrays in Bacillus subtilis using immunofluorescence and live cell fluorescence microscopy. We found that the receptors were localized in clusters at the poles in most cells. However, when the cells were exposed to attractant, the number exhibiting polar clusters was reduced roughly 2-fold, whereas the number exhibiting lateral clusters distinct from the poles increased significantly. These changes in receptor clustering were reversible as polar localization was reestablished in adapted cells. We also investigated the dynamic localization of CheV, a hybrid protein consisting of an N-terminal CheW-like adaptor domain and a C-terminal response regulator domain that is known to be phosphorylated by CheA, using immunofluorescence. Interestingly, we found that CheV was localized predominantly at lateral clusters in unstimulated cells. However, upon exposure to attractant, CheV was found to be predominantly localized to the cell poles. Moreover, changes in CheV localization are phosphorylation-dependent. Collectively, these results suggest that the chemotaxis signaling arrays in B. subtilis are dynamic structures and that feedback loops involving phosphorylation may regulate the positioning of individual proteins.     

Genetic evidence for interaction between the CheW and Tsr proteins during chemoreceptor signaling by Escherichia coli.

This study presents two lines of genetic evidence consistent with the premise that CheW, a cytoplasmic component of the chemotactic signaling system of Escherichia coli, interacts directly with Tsr, the membrane-bound serine chemoreceptor. (i) We demonstrated phenotypic suppression between 10 missense mutant CheW proteins and six missense mutant Tsr proteins. Most of these mutant proteins had leaky chemotaxis defects and were partially dominant, implying relatively minor functional alterations. Their suppression pattern was allele specific, suggesting that the mutant proteins have compensatory conformational changes at sites of interactive contact. (ii) We isolated five partially dominant CheW mutations and found that four of them were similar or identical to the suppressible CheW mutant proteins. This implies that there are only a few ways in which CheW function can be altered to produce dominant defects and that dominance is mediated through interactions of CheW with Tsr. The amino acid replacements in these mutant proteins were inferred from their DNA sequence changes. The CheW mutations were located in five regularly spaced clusters in the first two-thirds of the protein. The Tsr mutations were located in a highly conserved region in the middle of the cytoplasmic signaling domain. The hydrophobic moments, overall hydrophobicities, and predicted secondary structures of the mutant segments were consistent with the possibility that they are located at the surface of the CheW and Tsr molecules and represent the contact sites between these two proteins.     

A remote CheZ orthologue retains phosphatase function

Aspartyl‐phosphate phosphatases underlie the rapid responses of bacterial chemotaxis. One such phosphatase, CheZ, was originally proposed to be restricted to beta and gamma proteobacter, suggesting only a small subset of microbes relied on this protein. A putative CheZ phosphatase was identified genetically in the epsilon proteobacter Helicobacter pylori (Mol Micro 61:187). H. pylori utilizes a chemotaxis system consisting of CheAY, three CheVs, CheW, CheY_HP and the putative CheZ to colonize the host stomach. Here we investigate whether this CheZ has phosphatase activity. We phosphorylated potential targets in vitro using either a phosphodonor or the CheAY kinase and [γ‐³²P]‐ATP, and found that H. pylori CheZ (CheZ_HP) efficiently dephosphorylates CheY_HP and CheAY and has additional weak activity on CheV2. We detected no phosphatase activity towards CheV1 or CheV3. Mutations corresponding to Escherichia coli CheZ active site residues or deletion of the C‐terminal region inactivate CheZ_HP phosphatase activity, suggesting the two CheZs function similarly. Bioinformatics analysis suggests that CheZ phosphatases are found in all proteobacteria classes, as well as classes Aquificae, Deferribacteres, Nitrospira and Sphingobacteria, demonstrating that CheZ phosphatases are broadly distributed within Gram‐negative bacteria.     

CheA kinase and chemoreceptor interaction surfaces on CheW

Chemotactic responses of Escherichia coli to aspartic acid are initiated by a ternary protein complex composed of Tar (chemoreceptor), CheA (kinase), and CheW (a coupling protein that binds to both Tar and CheA and links their activities). We used a genetic selection based on the yeast two-hybrid assay to identify nine cheW point mutations that specifically disrupted CheW interaction with CheA but not with Tar. We sequenced these single point mutants and purified four of the mutant CheW proteins for detailed biochemical characterizations that demonstrated the weakened affinity of the mutant CheW proteins for CheA, but not for Tar. In the three-dimensional structure of CheW, the positions affected by these mutations cluster on one face of the protein, defining a potential binding interface for interaction of CheW with CheA. We used a similar two-hybrid approach to identify four mutation sites that disrupted CheW binding to Tar. Mapping of these "Tar-sensitive" mutation sites and those from previous suppressor analysis onto the structure of CheW defined an extended surface on a face of the protein that is adjacent to the CheA-binding surface and that may serve as an interface for CheW binding to Tar.     

Sequence and characterization of Bacillus subtilis CheW.

A Bacillus subtilis open reading frame (ORF) encoding a predicted polypeptide of 156 amino acids was subcloned and sequenced. The polypeptide was found to be homologous to CheW of Escherichia coli, sharing 28.6% amino acid identity. The ORF was verified by using a bacteriophage T7 expression system in E. coli. The gene was inactivated by insertion of a nonpolor chloramphenicol acetyltransferase cassette in its N-terminal region. In the absence of chemoeffectors, the mutant displayed a smooth swimming bias, with some tumbling. The CheW- mutant was defective on swarm plates but was complemented by a plasmid that expressed wild type CheW. Addition of attractant or repellent to the CheW- mutant resulted in transient smooth swimming or tumbling, respectively. However, capillary assays revealed that chemotaxis was substantially impaired in the mutant strain.     

Reconstitution of signaling in bacterial chemotaxis.

Strains missing several genes required for chemotaxis toward amino acids, peptides, and certain sugars were tethered and their rotational behavior was analyzed. Null strains (called gutted) were deleted for genes that code for the transducers Tsr, Tar, Tap, and Trg and for the cytoplasmic proteins CheA, CheW, CheR, CheB, CheY, and CheZ. Motor switch components were wild type, flaAII(cheC), or flaBII(cheV). Gutted cells with wild-type motors spun exclusively counterclockwise, while those with mutant motors changed their directions of rotation. CheY reduced the bias (the fraction of time that cells spun counterclockwise) in either case. CheZ offset the effect of CheY to an extent that varied with switch allele but did not change the bias when tested alone. Transducers also increased the bias in the presence of CheY but not when tested alone. However, cells containing transducers and CheY failed to respond to attractants or repellents normally detected in the periplasm. This sensitivity was restored by addition of CheA and CheW. Thus, CheY both enhances clockwise rotation and couples the transducers to the flagella. CheZ acts, at the level of the motor, as a CheY antagonist. CheA or CheW or both are required to complete the signal pathway. A model is presented that explains these results and is consistent with other data found in the literature.     

flaAII (motC, cheV) of Salmonella typhimurium is a structural gene involved in energization and switching of the flagellar motor.

The flaAII gene of Salmonella typhimurium has also been termed motC and cheV, because defective alleles may give rise to a nonflagellate, paralyzed, or nonchemotactic phenotype. We isolated a temperature-sensitive motility mutant (MY1) and have found that the mutation occurs in the flaAII gene. In temperature-jump experiments, MY1 could be converted from highly motile to paralyzed within 0.5 s, demonstrating that flaAII is a structural gene whose product is immediately essential for motor rotation. The mutant, although chemotactic at permissive temperatures (less than 36 degrees C), had a higher clockwise rotational bias than did the wild type; it can therefore be regarded simultaneously as motC(Ts) and cheV (tumbly). The only previously reported S. typhimurium cheV mutant was smooth-swimming. A shift toward counterclockwise bias accompanied loss of rotational speed in the restrictive temperature range. This result, by analogy with known proton motive force effects on motor switching, further indicates a central role of the flaAII (motC, cheV) protein in the energy transduction and switching process. Since there is no evidence associating it with the isolable entity known as the basal body, it may reside at the cytoplasmic face of the flagellar motor.     

Conformational Coupling between Receptor and Kinase Binding Sites through a Conserved Salt Bridge in a Signaling Complex Scaffold Protein

Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD), NMR spectroscopy, and circular dichroism (CD), we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.     

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1‐10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1^‐ isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l ‐aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild‐type C. jejuni cells and that of a tlp1^‐ isogenic mutant, specifically towards aspartate. Furthermore, using yeast two‐hybrid and three‐hybrid systems for analysis of protein–protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.     

CheW binding interactions with CheA and Tar. Importance for chemotaxis signaling in Escherichia coli

The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated. To better define the role of CheW and its binding interactions, we performed biochemical characterization of six mutant variants of CheW. We examined the ability of the purified mutant CheW proteins to bind to CheA and Tar, to promote formation of active ternary complexes, and to support chemotaxis in vivo. Our results indicate that mutations which eliminate CheW binding to Tar (V36M) or to CheA (G57D) result in a complete inability to form active ternary complexes in vitro and render the CheW protein incapable of mediating chemotaxis in vivo. The in vivo signaling pathway can, however, tolerate moderate changes in CheW-Tar and CheW-CheA affinities observed with several of the mutants (G133E, G41D, and 154ocr). One mutant (R62H) provided surprising results that may indicate a role for CheW in addition to binding CheA/receptors and promoting ternary complex formation.     

Role of the CheW protein in bacterial chemotaxis: overexpression is equivalent to absence.

The cheW gene from Escherichia coli has been cloned an inducible promoter, and the effects of the overproduction of the CheW protein on chemotactic behavior and receptor covalent modification have been examined. Plasmids that contain the cheW gene behind a regulatable promoter complement a cheW mutation when the CheW protein is produced at low levels. However, when the CheW protein is greatly overproduced in either a wild-type strain or a cheW mutant, chemotaxis is greatly inhibited, cheW null mutant cells swim smoothly as if they were constantly responding to an attractant. Surprisingly, cells in which the CheW protein is overproduced also swim smoothly. The behavioral defect produced by overproduction of the CheW protein does not require the presence of the cheR, cheB, or cheZ gene. Receptor demethylation is also inhibited by overproduction of the CheW protein, as it is by a mutation in the cheW gene or a response to an attractant. In all respects, therefore, overproduction of the CheW protein has the same consequences as does a mutation in the cheW gene or a response to an attractant. A model involving two states of the CheW protein is proposed to explain its role in bacterial chemotaxis.     

Different evolutionary constraints on chemotaxis proteins CheW and CheY revealed by heterologous expression studies and protein sequence analysis

CheW and CheY are single-domain proteins from a signal transduction pathway that transmits information from transmembrane receptors to flagellar motors in bacterial chemotaxis. In various bacterial and archaeal species, the cheW and cheY genes are usually encoded within homologous chemotaxis operons. We examined evolutionary changes in these two proteins from distantly related proteobacterial species, Escherichia coli and Azospirillum brasilense. We analyzed the functions of divergent CheW and CheY proteins from A. brasilense by heterologous expression in E. coli wild-type and mutant strains. Both proteins were able to specifically inhibit chemotaxis of a wild-type E. coli strain; however, only CheW from A. brasilense was able to restore signal transduction in a corresponding mutant of E. coli. Detailed protein sequence analysis of CheW and CheY homologs from the two species revealed substantial differences in the types of amino acid substitutions in the two proteins. Multiple, but conservative, substitutions were found in CheW homologs. No severe mismatches were found between the CheW homologs in positions that are known to be structurally or functionally important. Substitutions in CheY homologs were found to be less conservative and occurred in positions that are critical for interactions with other components of the signal transduction pathway. Our findings suggest that proteins from the same cellular pathway encoded by genes from the same operon have different evolutionary constraints on their structures that reflect differences in their functions.     

Proteomic mapping of a suppressor of non-chemotactic cheW mutants reveals that Helicobacter pylori contains a new chemotaxis protein

Bacterial chemotaxis is a colonization factor for the ulcer-causing pathogen Helicobacter pylori. H. pylori contains genes encoding the chemotaxis signalling proteins CheW, CheA and CheY; CheW couples chemoreceptors to the CheA kinase and is essential for chemotaxis. While characterizing a cheW mutant, we isolated a spontaneous, chemotactic variant (Che+). We determined that this phenotype was caused by a genetic change unlinked to the original cheW mutation. To locate the underlying Che+ mutation, we compared total protein profiles of the non-chemotactic mutant (cheW) with those from the cheW Che+ variant by two-dimensional differential in-gel electrophoresis. One protein was found only in the cheW Che+ variant. This protein was identified by MS/MS as HP0170, a hypothetical protein with no known function. DNA sequencing verified that hp0170 was mutated in the cheW Che+ suppressor, and deletion of this open reading frame in the cheW background nearly recapitulated the Che+ suppressor phenotype. Using hidden Markov models, we found that HP0170 is a remote homologue of E. coli CheZ. CheZ interacts with phosphorylated CheY and stimulates its autodephosphorylation. CheZ was not predicted to be present in epsilon-proteobacteria. We found that chemotaxis in the cheW Che+ suppressor depended on both cheY and cheA. We hypothesize that a small amount of phosphorylated CheY is generated via CheA in the cheW mutant, and this amount is sufficient to affect flagellar rotation when HP0170 is removed. Our results suggest that HP0170 is a remote homologue of CheZ, and that CheZ homologues are found in a broader range of bacteria than previously supposed.     

RecA Protein Plays a Role in the Chemotactic Response and Chemoreceptor Clustering of Salmonella enterica

The RecA protein is the main bacterial recombinase and the activator of the SOS system. In Escherichia coli and Salmonella enterica sv. Typhimurium, RecA is also essential for swarming, a flagellar-driven surface translocation mechanism widespread among bacteria. In this work, the direct interaction between RecA and the CheW coupling protein was confirmed, and the motility and chemotactic phenotype of a S. Typhimurium ΔrecA mutant was characterized through microfluidics, optical trapping, and quantitative capillary assays. The results demonstrate the tight association of RecA with the chemotaxis pathway and also its involvement in polar chemoreceptor cluster formation. RecA is therefore necessary for standard flagellar rotation switching, implying its essential role not only in swarming motility but also in the normal chemotactic response of S. Typhimurium.     

Mutational analysis of the chemoreceptor-coupling domain of the Escherichia coli chemotaxis signaling kinase CheA

During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and protein stability tests and in vitro assays with purified mutant proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/P3' interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA.     

Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway

The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 x CheA, 3 x CheW and 3 x CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild-type E. coli (RP437) by increasing its smooth-swimming bias. While some R. sphaeroides proteins restore tumbling to smooth-swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.     

Mutational analysis of N381, a key trimer contact residue in Tsr, the Escherichia coli serine chemoreceptor

Chemoreceptors such as Tsr, the serine receptor, function in trimer-of-dimer associations to mediate chemotactic behavior in Escherichia coli. The two subunits of each receptor homodimer occupy different positions in the trimer, one at its central axis and the other at the trimer periphery. Residue N381 of Tsr contributes to trimer stability through interactions with its counterparts in a central cavity surrounded by hydrophobic residues at the trimer axis. To assess the functional role of N381, we created and characterized a full set of amino acid replacements at this Tsr residue. We found that every amino acid replacement at N381 destroyed Tsr function, and all but one (N381G) of the mutant receptors also blocked signaling by Tar, the aspartate chemoreceptor. Tar jamming reflects the formation of signaling-defective mixed trimers of dimers, and in vivo assays with a trifunctional cross-linking reagent demonstrated trimer-based interactions between Tar and Tsr-N381 mutants. Mutant Tsr molecules with a charged amino acid or proline replacement exhibited the most severe trimer formation defects. These trimer-defective receptors, as well as most of the trimer-competent mutant receptors, were unable to form ternary signaling complexes with the CheA kinase and with CheW, which couples CheA to receptor control. Some of the trimer-competent mutant receptors, particularly those with a hydrophobic amino acid replacement, may not bind CheW/CheA because they form conformationally frozen or distorted trimers. These findings indicate that trimer dynamics probably are important for ternary complex assembly and that N381 may not be a direct binding determinant for CheW/CheA at the trimer periphery.