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Dual Recognition of the Bacterial Chemoreceptor by Chemotaxis-specific Domains of the CheR Methyltransferase*

Open AccessPublished:July 05, 2002DOI:https://doi.org/10.1074/jbc.M202001200
      Adaptation to persisting stimulation is required for highly sensitive detection of temporal changes of stimuli, and often involves covalent modification of receptors. Therefore, it is of vital importance to understand how a receptor and its cognate modifying enzyme(s) modulate each other through specific protein-protein interactions. In the chemotaxis of Escherichia coli, adaptation requires methylation of chemoreceptors (e.g.Tar) catalyzed by the CheR methyltransferase. CheR binds to the C-terminal NWETF sequence of a chemoreceptor that is distinct from the methylation sites. However, little is known about how CheR recognizes its methylation sites or how it is distributed in a cell. In this study, we used comparative genomics to demonstrate that the CheR chemotaxis methyltransferase contains three structurally and functionally distinct modules: (i) the catalytic domain common to a methyltransferase superfamily; (ii) the N-terminal domain; and (iii) the β-subdomain of the catalytic domain, both of which are found exclusively in chemotaxis methyltransferases. The only evolutionary conserved motif specific to CheR is the positively charged face of helix α2 in the N-terminal domain. The disulfide cross-linking analysis suggested that this face interacts with the methylation helix of Tar. We also demonstrated that CheR localizes to receptor clusters at cell poles via interaction of the β-subdomain with the NWETF sequence. Thus, the two chemotaxis-specific modules of CheR interact with distinct regions of the chemoreceptor for targeting to the receptor cluster and for recognition of the substrate sites, respectively.
      MCP
      methyl-accepting chemotaxis protein
      AdoMet
      S-adenosylmethionine
      GFP
      green fluorescent protein
      MBP
      maltose-binding protein
      MH
      methylation helix
      MTase
      methyltransferase
      In many sensory systems, transmembrane receptors recognize extracellular stimuli and transduce them into cytoplasmic signals to trigger defined physiological responses. These initial responses often diminish during persisting stimulation. The latter process, termed adaptation or desensitization, is essential for the detection of temporal changes of stimuli and/or the highly sensitive detection of stimuli over a comprehensive range. Covalent modifications of a receptor are often required for adaptation. In such cases, it is of vital importance to understand a regulated interplay between a receptor and modifying enzyme(s), including their mutual recognition and their subcellular localization, that assures spatially and temporally organized information processing.
      Molecular mechanisms of adaptation have been well characterized in the chemotaxis of Escherichia coli and Salmonella typhimurium (
      • Manson M.D.
      ,
      • Parkinson J.S.
      ,
      • Blair D.F.
      ,
      • Stock J.B.
      • Surette M.G.
      ,
      • Falke J.J.
      • Bass R.B.
      • Butler S.L.
      • Chervitz S.A.
      • Danielson M.A.
      ,
      • Armitage J.P.
      ). The transmembrane chemoreceptors, also known as the methyl-accepting chemotaxis proteins (MCPs),1 are methylated by the S-adenosylmethionine (AdoMet)-dependent methyltransferase (MTase) CheR and demethylated by the methylesterase CheB. In the resting state, an MCP is in equilibrium between methylation and demethylation. An attractant shifts the equilibrium toward methylation and a repellent toward demethylation. Each MCP has 4–5 glutamate residues located in two separate α helices (the first and second methylation helices (MH1 and MH2)) in the cytoplasmic domain (
      • Bass R.B.
      • Falke J.J.
      ,
      • Kim K.K.
      • Yokota H.
      • Kim S.-H.
      ). CheR has to recognize these residues (i.e. the substrate sites), but this interaction between CheR and an MCP has not been detected to date probably because it is weak and/or transient.
      In contrast, binding of CheR to the C-terminal pentapeptide sequence (NWETF) of the two high abundance receptors, the serine chemoreceptor Tsr and the aspartate chemoreceptor Tar, has been well characterized (
      • Wu J., Li, J., Li, G.
      • Long D.G.
      • Weis R.M.
      ,
      • Okumura H.
      • Nishiyama S.
      • Sasaki A.
      • Homma M.
      • Kawagishi I.
      ,
      • Djordjevic S.
      • Stock A.M.
      ,
      • Shiomi D.
      • Okumura H.
      • Homma M.
      • Kawagishi I.
      ). However, the low abundance chemoreceptors (the ribose-galactose transducer Trg and the dipeptide transducer Tap) do not have the sequence, indicating that the binding of CheR to the NWETF sequence might not be essential for its catalytic activity itself.
      The three-dimensional structure of CheR of S. typhimuriumrevealed that the monomeric protein consists of two domains (the N-terminal domain with no assigned function and the MTase domain) and one subdomain (the β-subdomain) (
      • Djordjevic S.
      • Stock A.M.
      ). The co-crystal of CheR and the pentapeptide revealed that the β-subdomain binds to the NWETF sequence (
      • Djordjevic S.
      • Stock A.M.
      ). Mutagenesis of the NWETF sequence of Tar demonstrated that it binds to CheR mainly through hydrophobic interaction (
      • Shiomi D.
      • Okumura H.
      • Homma M.
      • Kawagishi I.
      ). However, it is not clear how the CheR molecule is oriented when it binds to a chemoreceptor, nor how the NWETF sequence is oriented relative to the other part of the chemoreceptor molecule.
      Recently, subcellular localization of some proteins involved in chemotactic signal transduction (MCPs and Che proteins) has been studied using immunoelectron and immunofluorescence microscopy and YFP fusion proteins (
      • Maddock J.R.
      • Shapiro L.
      ,
      • Lybarger S.R.
      • Maddock J.R.
      ,
      • Lybarger S.R.
      • Maddock J.R.
      ,
      • Skidmore J.M.
      • Ellefson D.D.
      • McNamara B.P.
      • Couto M.M.
      • Wolfe A.J.
      • Maddock J.R.
      ,
      • Sourjik V.
      • Berg H.C.
      ). These studies demonstrated that MCPs cluster with the histidine kinase CheA and the adaptor protein CheW at cell poles. The localization and the clustering depend, at least to some extent, on CheA and CheW, but not on CheR or CheB. The localization and clustering of the chemotactic machinery at cell poles are proposed to be essential for amplification of input signals and for efficient methylation. The latter hypothesis assumes a high local concentration of CheR around the receptor/kinase cluster to provide a molecular basis of efficient methylation of both high abundance and low abundance receptors. Previous studies (
      • Okumura H.
      • Nishiyama S.
      • Sasaki A.
      • Homma M.
      • Kawagishi I.
      ,
      • Shiomi D.
      • Okumura H.
      • Homma M.
      • Kawagishi I.
      ,
      • Li J., Li, G.
      • Weis R.M.
      ,
      • Le Moual H.
      • Koshland Jr., D.E.
      ,
      • Feng X.
      • Lilly A.A.
      • Hazelbauer G.L.
      ) suggest that the NWETF sequence may serve to concentrate CheR around MCPs, but no direct evidence has been obtained.
      Moreover, the information about the structure-function relation of CheR was limited although the three-dimensional structure of S. typhimurium CheR has been determined in the absence and presence of the NWETF peptide (
      • Djordjevic S.
      • Stock A.M.
      ,
      • Djordjevic S.
      • Stock A.M.
      ) and the mutagenesis of the cysteine residues of S. typhimurium CheR (
      • Subbaramaiah K.
      • Charles H.
      • Simms S.A.
      ) was carried out. In this study, we took advantage of comparative genomic analysis to identify evolutionary conserved and therefore structurally and functionally important residues in the CheR protein family. Mutagenesis of some conserved residues in E. coli CheR demonstrated that some of them are functionally important. Characterization of GFP-CheR revealed that CheR localizes to cell poles through the interaction between its β-subdomain and the NWETF sequence of the chemoreceptor. Disulfide cross-linking assay was employed to examine the interaction between CheR and Tar and demonstrated that the positively charged residues in helix α2 of CheR are involved in the recognition of MH1. Thus, CheR interacts with the chemoreceptor through two distinct chemotaxis-specific modules to achieve efficient adaptation.

      DISCUSSION

      In this study, we examined how the MTase CheR interacts with its substrate, i.e. the chemoreceptor (MCP). More specifically, we asked how CheR is targeted to the receptor/kinase clusters at cell poles and how CheR recognizes the methylation sites of the chemoreceptor. We found that these two processes result from the distinct functions of CheR that reside in its two distinct domains.
      Comparative protein sequence analysis of CheR homologs and related proteins revealed that all of the CheR proteins have two domains and one subdomain: the CheR-specific N-terminal domain, the catalytic domain, and the β-subdomain. This organization is consistent with the crystallography of S. typhimurium CheR, suggesting that all of the CheR proteins share a common three-dimensional structure. It is also possible that they share common basic mechanisms for catalysis and substrate recognition. However, MCPs of many bacteria lack the C-terminal NWETF-type sequence that serves as a primary binding site of CheR in E. coli and S. typhimurium although all of the CheR proteins have the β-subdomain, which binds to the NWETF sequence in the case of S. typhimurium and E. coli CheR (Ref.
      • Djordjevic S.
      • Stock A.M.
      and this study). The β-subdomain can be divided into two groups (
      • Djordjevic S.
      • Stock A.M.
      ) (Fig. 2 B): longer β-loops (e.g. E. coli, S. typhimurium, andS. meliloti) and shorter β-loops (e.g. Vibrio cholerae and Bacillus subtilis). The CheR proteins of the bacteria whose MCPs contain the C-terminal NWETF-like motif belong to the former group. This difference in the length of the β-loop might reflect the differences in the mode of receptor recognition by CheR.
      In contrast to the recognition of the C-terminal tail of MCPs, little was known about the recognition of the methylation sites of MCPs by CheR. X-ray crystallography raised a possibility that the positively charged face of helix α2 of CheR might be involved in the interaction with MCPs (
      • Djordjevic S.
      • Stock A.M.
      ). However, such an interaction had not been detected biochemically. Here, the mutagenesis and the disulfide cross-linking analyses indicated that the positively charged face in helix α2 of CheR is involved in the recognition of MH1 of Tar. This is the first direct demonstration of the interaction between CheR and a methylation helix of any MCP. Among the residues tested, Arg-53 seems to be the most important residue for the recognition of MH1. This residue is strictly conserved among all of the CheR proteins except for theCampylobacter jejuni homolog, in which the corresponding residue is Lys.
      Thus, effective methylation requires two types of interaction between CheR and MCPs: one between the β-subdomain and the NWETF sequence (for the targeting of CheR to cell poles) and the other between the positively charged face in helix α2 and the negatively charged face of an MH (for the recognition of substrate sites) (Fig.7). An E. coli cell expresses some 5,000 monomers of MCPs and only several hundred molecules of CheR (
      • Simms S.A.
      • Stock A.M.
      • Stock J.B.
      ). Therefore, the targeting of CheR to the C-terminal tail of MCPs may be required to concentrate CheR molecules around receptor/kinase clusters at cell poles. Increased probability of CheR to collide with MCP molecules may then allow it to interact with the negatively charged face of an MH. This interaction between helix α2 and an MH is predicted to be weak and/or transient. CheR might slide on the negatively charged face of MH1, which contains three methylation sites with intervals of two turns of the helix, to monitor the methylation sites.
      Figure thumbnail gr7
      Figure 7Two modes of interaction between CheR (left) and MCP (right). The β-subdomain and the α2 helix of CheR interact with the C-terminal pentapeptide (NWETF) sequence and the MHs, respectively. The CheR structure (green) determined in the presence of the pentapeptide (blue) (
      • Djordjevic S.
      • Stock A.M.
      ) is shown with the crystal structure of the cytoplasmic fragment of Tsr (
      • Kim K.K.
      • Yokota H.
      • Kim S.-H.
      ) by placing the α2 helix of CheR to face the first three methylation sites (violet) of one subunit (blue) of the Tsr dimer. The other subunit of Tsr is shown in gray. The NWETF sequence is tentatively connected to the main part of the cytoplasmic domain with abroken line, although it is not clear whether CheR can bind simultaneously to these two parts of the MCP. CheR residues involved in receptor recognition and catalysis are shown in red andyellow, respectively.
      It is still unknown how CheR is oriented when it binds to an MCP. The binding of the NWETF sequence to the β-subdomain was visualized by x-ray crystallography (
      • Djordjevic S.
      • Stock A.M.
      ). However, it is unclear how the rest of the MCP molecule is oriented and whether CheR can catalyze methylation of an MCP molecule while it is anchored to the C-terminal tail of the same molecule or the partner subunit of the same dimer. It was also suggested that CheR can catalyze methylation of a neighboring MCP molecule within an MCP cluster (
      • Li J., Li, G.
      • Weis R.M.
      ,
      • Le Moual H.
      • Koshland Jr., D.E.
      ). This interdimer methylation explains why a low abundance MCP can be methylated efficiently in the presence of a high abundance MCP (
      • Yamamoto K.
      • Macnab R.M.
      • Imae Y.
      ). Again, it is not clear whether this can be achieved without dissociation of CheR from the NWETF sequence. In any case, the simultaneous anchoring and catalysis would require large flexibility of the MCP molecule. Because the affinity of CheR for the pentapeptide is not very high, it is also possible that CheR is dissociated from the NWETF sequence during catalysis.
      We also examined subcellular localization of CheR. GFP-CheR localized to cell poles only in the presence of an MCP with the NWETF sequence. The mutagenesis of the CheR part suggested that the targeting of CheR to cell poles depends primarily on the interaction between CheR and the NWETF sequence. These results are consistent with the hypothesis that the NWETF sequence serves to concentrate CheR around MCPs at cell poles.
      It should be noted that the abilities of the wild-type and mutant versions of GFP-CheR to localize to cell poles (Fig. 4, right panels) appeared to vary: wild-type > R53A > D154A. In the experimental conditions applied, the GFP-CheR proteins were mildly overproduced relative to chromosome-encoded CheR. Therefore, the methylation level of Tar would be different from one strain to another. This may suggest two possibilities: the methylation levels of MCPs might affect the subcellular localization of: (i) CheR and/or (ii) MCPs themselves. The high abundance MCPs (Tar and Tsr) localize to cell poles and form clusters with CheA and CheW, whereas the low abundance MCPs (Tap and Trg) also localize to cell poles but do not form a cluster (
      • Lybarger S.R.
      • Maddock J.R.
      ). The latter receptors are poor substrates for CheR because they lack the C-terminal NWETF sequence. Taken together, it is possible that the methylation levels of MCPs would be critical for their clustering at cell poles. However, the polar localization of the high abundance MCPs does not seem to require CheR and CheB (
      • Lybarger S.R.
      • Maddock J.R.
      ). Further analyses are required to clarify this issue.
      The Cys-substituted CheR proteins were more effectively cross-linked to Tar-EEEE than to Tar-QQQQ. This may result from the electrostatic nature of the interaction between MHs of MCPs and helix α2 of CheR. It is also possible that receptor amidation (and hence methylation) alters the conformation of the MHs to reduce its affinity to helix α2 of CheR. In any case, this finding is consistent with the notion that upon methylation, an MCP becomes a poorer substrate of CheR.
      CheB also binds to the C-terminal NWETF sequence of MCPs (
      • Barnakov A.N.
      • Barnakov L.A.
      • Hazelbauer G.L.
      ,
      • Barnakov A.N.
      • Barnakova L.A.
      • Hazelbauer G.L.
      ) and has to recognize MHs of MCPs. However, CheB may be different from CheR in these respects. The affinity of CheB for the pentapeptide is much lower than that of CheR and the cellular concentration of CheB is much higher than that of CheR. Therefore, it is hard to imagine that the NWETF sequence serves to recruit CheB around MCPs. As for the substrate recognition, CheB recognizes methylated Glu residues to hydrolyze them, whereas CheR recognizes unmethylated Glu residues. Consistent with this consideration, E. coli CheB does not have a positively charged cluster in the primary sequence. Thus, it is intriguing to compare the mechanisms of receptor recognition of CheB with those of CheR.

      Acknowledgments

      We thank Drs. J. Beckwith and J. S. Parkinson for providing plasmids and bacterial strains. We acknowledge the following genome sequencing centers (and their funding agencies) for providing access to incomplete genome data: the Sanger Centre (Beowulf Genomics), the Joint Genome Institute (United States Department of Energy), the Institute for Genome Research (United States Department of Energy), and the University of Oklahoma (National Science Foundation).