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Location of the Receptor-interaction Site on CheB, the Methylesterase Response Regulator of Bacterial Chemotaxis

2001; Elsevier BV; Volume: 276; Issue: 35 Linguagem: Inglês

10.1074/jbc.m105925200

1083-351X

Alexander N. Barnakov, Ludmila A. Barnakova, Gerald L. Hazelbauer,

Antibiotic Resistance in Bacteria

Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors, specifically methylation and demethylation of glutamates catalyzed by methyltransferase CheR and methylesterase CheB. The methylesterase is a two-domain response regulator in which phosphorylation of the regulatory domain enhances activity of the catalytic domain. InEscherichia coli and Salmonella typhimurium, a crucial determinant of efficient methylation and demethylation is a specific pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from sites of enzymatic action. Each enzyme binds pentapeptide, but the site of binding has been located only for CheR. Here we locate the pentapeptide-binding site on CheB by assessing catalytic activity and pentapeptide binding of CheB fragments, protection of CheB from proteolysis by pentapeptide, and interference with pentapeptide-CheB interaction by a CheB segment. The results place the binding site near the hinge between regulatory and catalytic domains, in a segment spanning the carboxyl-terminal end of the regulatory domain and the beginning of the linker that stretches to the catalytic domain. This location is quite different from the catalytic domain location of the pentapeptide-binding site on CheR and is likely to reflect the rather different ways in which pentapeptide binding enhances enzymatic action for the methyltransferase and the methylesterase. Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors, specifically methylation and demethylation of glutamates catalyzed by methyltransferase CheR and methylesterase CheB. The methylesterase is a two-domain response regulator in which phosphorylation of the regulatory domain enhances activity of the catalytic domain. InEscherichia coli and Salmonella typhimurium, a crucial determinant of efficient methylation and demethylation is a specific pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from sites of enzymatic action. Each enzyme binds pentapeptide, but the site of binding has been located only for CheR. Here we locate the pentapeptide-binding site on CheB by assessing catalytic activity and pentapeptide binding of CheB fragments, protection of CheB from proteolysis by pentapeptide, and interference with pentapeptide-CheB interaction by a CheB segment. The results place the binding site near the hinge between regulatory and catalytic domains, in a segment spanning the carboxyl-terminal end of the regulatory domain and the beginning of the linker that stretches to the catalytic domain. This location is quite different from the catalytic domain location of the pentapeptide-binding site on CheR and is likely to reflect the rather different ways in which pentapeptide binding enhances enzymatic action for the methyltransferase and the methylesterase. Tar missing its final five amino acids the isolated catalytic domain of CheB 50 mm Tris-HCl, pH 7.5, 0.5 mm EDTA, 2 mm dithiothreitol, 10% glycerol 50 mm HEPES, pH 7.5, 0.5 mm EDTA, 10% (w/w) glycerol polyacrylamide gel electrophoresis The mechanistic basis of sensory adaptation and gradient sensing in bacterial chemotaxis is reversible covalent modification of chemoreceptors (1Springer M.S. Goy M.F. Adler J. Nature. 1979; 280: 279-284Crossref PubMed Scopus (246) Google Scholar, 2Hazelbauer G.L. Park C. Nowlin D.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1448-1452Crossref PubMed Scopus (40) Google Scholar). Specific glutamyl residues in the cytoplasmic domain of chemoreceptors are methylated to form carboxyl methylesters and demethylated to reform the carboxyl groups (3Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar). The reactions are catalyzed by enzymes specific for the chemosensory system, the methyltransferase CheR (4Springer W.R. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 533-537Crossref PubMed Scopus (187) Google Scholar) and the methylesterase CheB (5Stock J.B. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3659-3663Crossref PubMed Scopus (141) Google Scholar). In the well studied chemosensory systems of Escherichia coliand Salmonella typhimurium (see Refs. 6Falke J.J. Bass R.B. Butler S.L. Chervitz S.A. Danielson M.A. Annu. Rev. Cell Dev. Biol. 1997; 13: 457-512Crossref PubMed Scopus (420) Google Scholar and 7Hazelbauer G.L. Adelman G. Smith B.H. Encyclopedia of Neuroscience. Elsevier Science Publishing Co., Inc., New York1999: 181-183Google Scholar for recent reviews), chemoreceptors have four to six methyl-accepting glutamyl residues, four of which are at conserved positions (3Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar, 8Nowlin D.M. Bollinger J. Hazelbauer G.L. J. Biol. Chem. 1987; 262: 6039-6045Abstract Full Text PDF PubMed Google Scholar, 9Nowlin D.M. Bollinger J. Hazelbauer G.L. Proteins. 1988; 3: 102-112Crossref PubMed Scopus (27) Google Scholar, 10Terwilliger T.C. Koshland Jr., D.E. J. Biol. Chem. 1984; 259: 7719-7725Abstract Full Text PDF PubMed Google Scholar, 11Rice M.S. Dahlquist F.W. J. Biol. Chem. 1991; 266: 9746-9753Abstract Full Text PDF PubMed Google Scholar). Two of the conserved sites are created by deamidation of glutaminyl residues in a reaction catalyzed by CheB, the same enzyme that catalyzes demethylation (3Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar). Thus CheB is both a methylesterase and a deamidase.Central to the chemosensory system are complexes consisting of chemoreceptor, the autophosporylating histidine kinase, CheA, and the SH3-related coupling protein, CheW (12Gegner J.A. Graham D.R. Roth A.F. Dahlquist F.W. Cell. 1992; 70: 975-982Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 13Schuster S.C. Swanson R.V. Alex L.A. Bourret R.B. Simon M.I. Nature. 1993; 365: 343-347Crossref PubMed Scopus (229) Google Scholar). Interaction of kinase with receptor in the complex substantially increases the otherwise low rate of kinase autophosphorylation (14Borkovich K.A. Kaplan N. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1208-1212Crossref PubMed Scopus (237) Google Scholar). The phosphoryl group on the phosphohistidine can be transferred to an aspartyl residue on either of two response regulators, the single domain protein CheY and the two-domain enzyme CheB (15Hess J.F. Oosawa K. Kaplan N. Simon M.I. Cell. 1988; 53: 79-87Abstract Full Text PDF PubMed Scopus (395) Google Scholar). Phospho-CheY binds the flagellar rotary motor, switching rotation from the default counterclockwise direction to clockwise (16Welch M. Oosawa K. Aizawa S. Eisenbach M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8787-8791Crossref PubMed Scopus (348) Google Scholar). CheB is phosphorylated on a regulatory domain that is structurally homologous to CheY (17Lupas A. Stock J. J. Biol. Chem. 1989; 264: 17337-17342Abstract Full Text PDF PubMed Google Scholar). This phosphorylation activates the second domain of the enzyme (17Lupas A. Stock J. J. Biol. Chem. 1989; 264: 17337-17342Abstract Full Text PDF PubMed Google Scholar) that catalyzes methyl ester and amide hydrolysis. Binding of chemoattractant to receptor lowers the activity of the associated kinase, reducing levels of the phosphorylated CheY and CheB (14Borkovich K.A. Kaplan N. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1208-1212Crossref PubMed Scopus (237) Google Scholar, 18Ninfa E.G. Stock A. Mowbray S. Stock J. J. Biol. Chem. 1991; 266: 9764-9770Abstract Full Text PDF PubMed Google Scholar). Reduced cellular levels of phospho-CheY shift the flagellar rotational bias and alter the pattern of swimming (19Barak R. Welch M. Yanovsky A. Oosawa K. Eisenbach M. Biochemistry. 1992; 31: 10099-10107Crossref PubMed Scopus (65) Google Scholar). However, these changes are transient, because attractant binding also sets in motion the feedback loop of sensory adaptation that re-establishes the pre-stimulus rotational bias and swimming pattern. In this process an increase in receptor methylation creates a compensatory change in the receptor-kinase complex that restores CheA activity to its null, receptor-activated state (20Borkovich K.A. Alex L.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6756-6760Crossref PubMed Scopus (157) Google Scholar) even though the increased level of attractant persists.What factors are important for efficient adaptational modification? The side chains that are methylated and demethylated (some of which were glutamines deamidated to create methyl-accepting sites) are spaced seven apart in the receptor sequence (3Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar), are on solvent-exposed surfaces of the helices of the chemoreceptor cytoplasmic domain (21Kim K.K. Yokota H. Kim S.H. Nature. 1999; 400: 787-792Crossref PubMed Scopus (384) Google Scholar), and are bracketed by sequences that share common features and influence kinetic preferences among sites (8Nowlin D.M. Bollinger J. Hazelbauer G.L. J. Biol. Chem. 1987; 262: 6039-6045Abstract Full Text PDF PubMed Google Scholar, 9Nowlin D.M. Bollinger J. Hazelbauer G.L. Proteins. 1988; 3: 102-112Crossref PubMed Scopus (27) Google Scholar, 10Terwilliger T.C. Koshland Jr., D.E. J. Biol. Chem. 1984; 259: 7719-7725Abstract Full Text PDF PubMed Google Scholar, 22Terwilliger T.C. Wang J.Y. Koshland D.E. J. Biol. Chem. 1986; 261: 10814-10820Abstract Full Text PDF PubMed Google Scholar, 23Terwilliger T.C. Wang J.Y. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6707-6710Crossref PubMed Scopus (32) Google Scholar, 24Shapiro M.J. Chakrabarti I. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1053-1056Crossref PubMed Scopus (26) Google Scholar). However, a crucial determinant of efficient methylation, demethylation, and deamidation is distant from the sites of modification. This determinant, identified in studies of chemoreceptors in E. coli and S. typhimurium, is the presence at the chemoreceptor carboxyl terminus of a pentapeptide sequence, asparagine-tryptophan-glutamate-threonine-phenylalanine, NWETF in the one letter code (25Wu J. Li J. Li G. Long D.G. Weis R.M. Biochemistry. 1996; 35: 4984-4993Crossref PubMed Scopus (153) Google Scholar, 26Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar). Both the methyltransferase (25Wu J. Li J. Li G. Long D.G. Weis R.M. Biochemistry. 1996; 35: 4984-4993Crossref PubMed Scopus (153) Google Scholar) and the methylesterase (26Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar) bind to this sequence. Chemoreceptors lacking the pentapeptide naturally or as the result of engineered truncations or mutations are inefficiently methylated, demethylated, and deamidated (26Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar, 27Li J. Li G. Weis R.M. Biochemistry. 1997; 36: 11851-11857Crossref PubMed Scopus (72) Google Scholar, 28Le Moual H. Quang T. Koshland Jr., D.E. Biochemistry. 1997; 36: 13441-13448Crossref PubMed Scopus (55) Google Scholar, 29Okumura H. Nishiyama S. Sasaki A. Homma M. Kawagishi I. J. Bacteriol. 1998; 180: 1862-1868Crossref PubMed Google Scholar, 30Feng X. Lilly A.A. Hazelbauer G.L. J. Bacteriol. 1999; 181: 3164-3171Crossref PubMed Google Scholar) and are ineffective on their own at mediating tactic response and directed movement (30Feng X. Lilly A.A. Hazelbauer G.L. J. Bacteriol. 1999; 181: 3164-3171Crossref PubMed Google Scholar, 31Hazelbauer G.L. Engström P. Nature. 1980; 283: 98-100Crossref PubMed Scopus (45) Google Scholar, 32Yamamoto K. Macnab R.M. Imae Y. J. Bacteriol. 1990; 172: 383-388Crossref PubMed Google Scholar, 33Feng X. Baumgartner J.W. Hazelbauer G.L. J. Bacteriol. 1997; 179: 6714-6720Crossref PubMed Google Scholar, 34Weerasuriya S. Schneider B.M. Manson M.D. J. Bacteriol. 1998; 180: 914-920Crossref PubMed Google Scholar). Such receptors mediate effective taxis only with the assistance of NWETF-containing receptors (30Feng X. Lilly A.A. Hazelbauer G.L. J. Bacteriol. 1999; 181: 3164-3171Crossref PubMed Google Scholar, 31Hazelbauer G.L. Engström P. Nature. 1980; 283: 98-100Crossref PubMed Scopus (45) Google Scholar, 33Feng X. Baumgartner J.W. Hazelbauer G.L. J. Bacteriol. 1997; 179: 6714-6720Crossref PubMed Google Scholar,34Weerasuriya S. Schneider B.M. Manson M.D. J. Bacteriol. 1998; 180: 914-920Crossref PubMed Google Scholar).CheR has an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain that exhibit structural features conserved in the family of methyltransferases (35Djordjevic S. Stock A.M. Structure. 1997; 5: 545-558Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The binding site for the receptor pentapeptide is a topological insertion in the conserved fold of the catalytic domain, an insertion found specifically in chemotaxis-related methyltransferases (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). This inserted β-subdomain, containing a three-stranded β-sheet, binds the receptor pentapeptide by aligning it as a fourth strand of that β-sheet (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). In species other thanE. coli and S. typhimurium, binding of CheR to chemoreceptors has yet to be investigated, but the sequences of CheR β-subdomains and of receptor carboxyl termini co-vary from the sequences in E. coli in a way that implies a conserved interaction (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar).For the methylesterase CheB, less is known about the interaction of enzyme with the receptor pentapeptide. As a first step in learning more, we investigated the location of the NWETF-binding site. Like CheR, CheB has an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain (see Fig. 1). The catalytic domains of the two enzymes are structurally and presumably evolutionarily related (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar). Both are doubly wound α/β structures with a topological insertion. In CheR, this insertion is the β-sheet subdomain that binds the receptor pentapeptide (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). For CheB, the insertion is a β-hairpin positioned on the surface of the catalytic domain in the same relation to the enzyme active site as the pentapeptide-binding β-subdomain of CheR (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar). Thus the β-hairpin was a candidate for the pentapeptide-binding site in CheB. We investigated the location of the pentapeptide-binding site by experiments that tested catalytic activity and pentapeptide binding of CheB fragments, protection of CheB from proteolysis by pentapeptide, and interference with NWETF-CheB interaction by short CheB peptides.DISCUSSIONIn this study we used a combination of experimental approaches to locate the site on CheB at which this two-component response regulator and methylesterase/deamidase binds the pentapeptide present at the carboxyl-terminal of chemoreceptors. The results of these different approaches provided a consistent pattern that located the pentapeptide-binding site at the juncture between the carboxyl-terminal end of α-5, the final piece of secondary structure in the regulatory domain, and the linker that connects the regulatory and catalytic domains. This location is consistent with the lack of detectable interaction between pentapeptide and the catalytic domain of CheB, assessed by functional or binding assays. The location explains the binding of a CheB fragment containing α-5, linker and catalytic domain, the lack of binding by a related fragment containing only the linker and catalytic domain, the protection by free NWETF pentapeptide of a specific tryptic site at the boundary of α-5 and linker, and the interference with pentapeptide-CheB binding by an 11-residue segment of CheB that includes the boundary between α-5 and the linker.The experimentally determined location of the pentapeptide-binding site in CheB could not have been predicted by analogy with the structurally related methyltransferase CheR. In CheR, the receptor-binding site is provided by the β-subdomain, a topological insertion in the catalytic domain. In CheB, the analogous structural unit is a β-hairpin insertion in the catalytic domain (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). If the receptor-binding site were analogously placed on CheR and CheB, then the pentapeptide-binding site in CheB would be on the β-hairpin of the catalytic domain, not at the regulatory domain-linker boundary. Yet none of our experiments implicated the catalytic domain of CheB in receptor binding, but instead all the evidence pointed toward the carboxyl end of the regulatory domain.A difference in location of the receptor-binding site on CheR and CheB parallels other differences between interactions of the two modification enzymes with the receptor pentapeptide. In work to be reported elsewhere, we found that binding of CheB to the NWETF receptor sequence is substantially weaker than binding of CheR and that the interaction is likely to enhance enzyme action through effects on catalysis rather than enzyme recruitment. 2A. Barnakov, L. Barnakova, and G. L. Hazelbauer, manuscript in preparation. It is plausible that a binding site affecting catalysis would involve α-5 and the linker, because these parts of CheB participate in the interface between the regulatory and catalytic domain (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar, 43Hughes C.A. Mandell J.G. Anand G.S. Stock A.M. Komives E.A. J. Mol. Biol. 2001; 307: 967-976Crossref PubMed Scopus (53) Google Scholar), and interaction of those two domains is likely to limit enzyme action (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). Models of how the regulatory domain controls the activity of the catalytic domain suggest that in the low activity state access of substrate to the catalytic domain is restricted by the position of the regulatory domain (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar, 42Anand G.S. Goudreau P.N. Lewis J.K. Stock A.M. Protein Sci. 2000; 9: 898-906Crossref PubMed Scopus (16) Google Scholar). Yet phosphorylation of the regulatory domain has only subtle effects on the interface between the domains (43Hughes C.A. Mandell J.G. Anand G.S. Stock A.M. Komives E.A. J. Mol. Biol. 2001; 307: 967-976Crossref PubMed Scopus (53) Google Scholar), implying that phosphorylation-mediated enhancement of CheB action is not simply an extensive exposure of an otherwise occluded surface of the catalytic domain. Because pentapeptide-mediated enhancement of CheB action is independent of and additive with enhancement by regulatory domain phosphorylation (26Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar), it could be that pentapeptide binding near the hinge between the two domains creates a separation of the domains or affects the catalytic site directly in a way not caused by phosphorylation. There is no evidence that pentapeptide binding affects CheR catalysis. Instead all observations about enhancement of CheR action by pentapeptide can be explained by considering NWETF a high affinity docking site that recruits the methyltransferase to the region of its substrate methyl-accepting residues. Thus the difference in placement of the receptor-interaction sites on the two enzymes of adaptational modification parallels a difference in functional effects.The impressive sensitivity of the chemotactic system to very small changes in concentration of attractants indicates that signaling includes a means of creating significant gain, an amplification of small differences in receptor occupancy to produce detectable responses (44Mesibov R. Adler J. J. Bacteriol. 1972; 112: 315-326Crossref PubMed Google Scholar, 45Jasuja R. Keyoung J. Reid G.P. Trentham D.R. Khan S. Biophys. J. 1999; 76: 1706-1719Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The mechanism of this amplification is a subject of active research. A recent study implicates the enzymes of adaptational modification as crucial contributors to excitatory gain (46Kim C. Jackson M. Lux R. Khan S. J. Mol. Biol. 2001; 307: 119-135Crossref PubMed Scopus (46) Google Scholar). The authors of this study suggest that response sensitivity could be controlled by differential binding of the modification enzymes to distinct conformations of the chemoreceptors (46Kim C. Jackson M. Lux R. Khan S. J. Mol. Biol. 2001; 307: 119-135Crossref PubMed Scopus (46) Google Scholar). Knowledge of the sites of receptor interaction on CheB, as well as CheR, sets the stage for detailed investigation of this suggestion. The mechanistic basis of sensory adaptation and gradient sensing in bacterial chemotaxis is reversible covalent modification of chemoreceptors (1Springer M.S. Goy M.F. Adler J. Nature. 1979; 280: 279-284Crossref PubMed Scopus (246) Google Scholar, 2Hazelbauer G.L. Park C. Nowlin D.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1448-1452Crossref PubMed Scopus (40) Google Scholar). Specific glutamyl residues in the cytoplasmic domain of chemoreceptors are methylated to form carboxyl methylesters and demethylated to reform the carboxyl groups (3Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar). The reactions are catalyzed by enzymes specific for the chemosensory system, the methyltransferase CheR (4Springer W.R. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 533-537Crossref PubMed Scopus (187) Google Scholar) and the methylesterase CheB (5Stock J.B. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3659-3663Crossref PubMed Scopus (141) Google Scholar). In the well studied chemosensory systems of Escherichia coliand Salmonella typhimurium (see Refs. 6Falke J.J. Bass R.B. Butler S.L. Chervitz S.A. Danielson M.A. Annu. Rev. Cell Dev. Biol. 1997; 13: 457-512Crossref PubMed Scopus (420) Google Scholar and 7Hazelbauer G.L. Adelman G. Smith B.H. Encyclopedia of Neuroscience. Elsevier Science Publishing Co., Inc., New York1999: 181-183Google Scholar for recent reviews), chemoreceptors have four to six methyl-accepting glutamyl residues, four of which are at conserved positions (3Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar, 8Nowlin D.M. Bollinger J. Hazelbauer G.L. J. Biol. Chem. 1987; 262: 6039-6045Abstract Full Text PDF PubMed Google Scholar, 9Nowlin D.M. Bollinger J. Hazelbauer G.L. Proteins. 1988; 3: 102-112Crossref PubMed Scopus (27) Google Scholar, 10Terwilliger T.C. Koshland Jr., D.E. J. Biol. Chem. 1984; 259: 7719-7725Abstract Full Text PDF PubMed Google Scholar, 11Rice M.S. Dahlquist F.W. J. Biol. Chem. 1991; 266: 9746-9753Abstract Full Text PDF PubMed Google Scholar). Two of the conserved sites are created by deamidation of glutaminyl residues in a reaction catalyzed by CheB, the same enzyme that catalyzes demethylation (3Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar). Thus CheB is both a methylesterase and a deamidase. Central to the chemosensory system are complexes consisting of chemoreceptor, the autophosporylating histidine kinase, CheA, and the SH3-related coupling protein, CheW (12Gegner J.A. Graham D.R. Roth A.F. Dahlquist F.W. Cell. 1992; 70: 975-982Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 13Schuster S.C. Swanson R.V. Alex L.A. Bourret R.B. Simon M.I. Nature. 1993; 365: 343-347Crossref PubMed Scopus (229) Google Scholar). Interaction of kinase with receptor in the complex substantially increases the otherwise low rate of kinase autophosphorylation (14Borkovich K.A. Kaplan N. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1208-1212Crossref PubMed Scopus (237) Google Scholar). The phosphoryl group on the phosphohistidine can be transferred to an aspartyl residue on either of two response regulators, the single domain protein CheY and the two-domain enzyme CheB (15Hess J.F. Oosawa K. Kaplan N. Simon M.I. Cell. 1988; 53: 79-87Abstract Full Text PDF PubMed Scopus (395) Google Scholar). Phospho-CheY binds the flagellar rotary motor, switching rotation from the default counterclockwise direction to clockwise (16Welch M. Oosawa K. Aizawa S. Eisenbach M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8787-8791Crossref PubMed Scopus (348) Google Scholar). CheB is phosphorylated on a regulatory domain that is structurally homologous to CheY (17Lupas A. Stock J. J. Biol. Chem. 1989; 264: 17337-17342Abstract Full Text PDF PubMed Google Scholar). This phosphorylation activates the second domain of the enzyme (17Lupas A. Stock J. J. Biol. Chem. 1989; 264: 17337-17342Abstract Full Text PDF PubMed Google Scholar) that catalyzes methyl ester and amide hydrolysis. Binding of chemoattractant to receptor lowers the activity of the associated kinase, reducing levels of the phosphorylated CheY and CheB (14Borkovich K.A. Kaplan N. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1208-1212Crossref PubMed Scopus (237) Google Scholar, 18Ninfa E.G. Stock A. Mowbray S. Stock J. J. Biol. Chem. 1991; 266: 9764-9770Abstract Full Text PDF PubMed Google Scholar). Reduced cellular levels of phospho-CheY shift the flagellar rotational bias and alter the pattern of swimming (19Barak R. Welch M. Yanovsky A. Oosawa K. Eisenbach M. Biochemistry. 1992; 31: 10099-10107Crossref PubMed Scopus (65) Google Scholar). However, these changes are transient, because attractant binding also sets in motion the feedback loop of sensory adaptation that re-establishes the pre-stimulus rotational bias and swimming pattern. In this process an increase in receptor methylation creates a compensatory change in the receptor-kinase complex that restores CheA activity to its null, receptor-activated state (20Borkovich K.A. Alex L.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6756-6760Crossref PubMed Scopus (157) Google Scholar) even though the increased level of attractant persists. What factors are important for efficient adaptational modification? The side chains that are methylated and demethylated (some of which were glutamines deamidated to create methyl-accepting sites) are spaced seven apart in the receptor sequence (3Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar), are on solvent-exposed surfaces of the helices of the chemoreceptor cytoplasmic domain (21Kim K.K. Yokota H. Kim S.H. Nature. 1999; 400: 787-792Crossref PubMed Scopus (384) Google Scholar), and are bracketed by sequences that share common features and influence kinetic preferences among sites (8Nowlin D.M. Bollinger J. Hazelbauer G.L. J. Biol. Chem. 1987; 262: 6039-6045Abstract Full Text PDF PubMed Google Scholar, 9Nowlin D.M. Bollinger J. Hazelbauer G.L. Proteins. 1988; 3: 102-112Crossref PubMed Scopus (27) Google Scholar, 10Terwilliger T.C. Koshland Jr., D.E. J. Biol. Chem. 1984; 259: 7719-7725Abstract Full Text PDF PubMed Google Scholar, 22Terwilliger T.C. Wang J.Y. Koshland D.E. J. Biol. Chem. 1986; 261: 10814-10820Abstract Full Text PDF PubMed Google Scholar, 23Terwilliger T.C. Wang J.Y. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6707-6710Crossref PubMed Scopus (32) Google Scholar, 24Shapiro M.J. Chakrabarti I. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1053-1056Crossref PubMed Scopus (26) Google Scholar). However, a crucial determinant of efficient methylation, demethylation, and deamidation is distant from the sites of modification. This determinant, identified in studies of chemoreceptors in E. coli and S. typhimurium, is the presence at the chemoreceptor carboxyl terminus of a pentapeptide sequence, asparagine-tryptophan-glutamate-threonine-phenylalanine, NWETF in the one letter code (25Wu J. Li J. Li G. Long D.G. Weis R.M. Biochemistry. 1996; 35: 4984-4993Crossref PubMed Scopus (153) Google Scholar, 26Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar). Both the methyltransferase (25Wu J. Li J. Li G. Long D.G. Weis R.M. Biochemistry. 1996; 35: 4984-4993Crossref PubMed Scopus (153) Google Scholar) and the methylesterase (26Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar) bind to this sequence. Chemoreceptors lacking the pentapeptide naturally or as the result of engineered truncations or mutations are inefficiently methylated, demethylated, and deamidated (26Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar, 27Li J. Li G. Weis R.M. Biochemistry. 1997; 36: 11851-11857Crossref PubMed Scopus (72) Google Scholar, 28Le Moual H. Quang T. Koshland Jr., D.E. Biochemistry. 1997; 36: 13441-13448Crossref PubMed Scopus (55) Google Scholar, 29Okumura H. Nishiyama S. Sasaki A. Homma M. Kawagishi I. J. Bacteriol. 1998; 180: 1862-1868Crossref PubMed Google Scholar, 30Feng X. Lilly A.A. Hazelbauer G.L. J. Bacteriol. 1999; 181: 3164-3171Crossref PubMed Google Scholar) and are ineffective on their own at mediating tactic response and directed movement (30Feng X. Lilly A.A. Hazelbauer G.L. J. Bacteriol. 1999; 181: 3164-3171Crossref PubMed Google Scholar, 31Hazelbauer G.L. Engström P. Nature. 1980; 283: 98-100Crossref PubMed Scopus (45) Google Scholar, 32Yamamoto K. Macnab R.M. Imae Y. J. Bacteriol. 1990; 172: 383-388Crossref PubMed Google Scholar, 33Feng X. Baumgartner J.W. Hazelbauer G.L. J. Bacteriol. 1997; 179: 6714-6720Crossref PubMed Google Scholar, 34Weerasuriya S. Schneider B.M. Manson M.D. J. Bacteriol. 1998; 180: 914-920Crossref PubMed Google Scholar). Such receptors mediate effective taxis only with the assistance of NWETF-containing receptors (30Feng X. Lilly A.A. Hazelbauer G.L. J. Bacteriol. 1999; 181: 3164-3171Crossref PubMed Google Scholar, 31Hazelbauer G.L. Engström P. Nature. 1980; 283: 98-100Crossref PubMed Scopus (45) Google Scholar, 33Feng X. Baumgartner J.W. Hazelbauer G.L. J. Bacteriol. 1997; 179: 6714-6720Crossref PubMed Google Scholar,34Weerasuriya S. Schneider B.M. Manson M.D. J. Bacteriol. 1998; 180: 914-920Crossref PubMed Google Scholar). CheR has an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain that exhibit structural features conserved in the family of methyltransferases (35Djordjevic S. Stock A.M. Structure. 1997; 5: 545-558Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The binding site for the receptor pentapeptide is a topological insertion in the conserved fold of the catalytic domain, an insertion found specifically in chemotaxis-related methyltransferases (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). This inserted β-subdomain, containing a three-stranded β-sheet, binds the receptor pentapeptide by aligning it as a fourth strand of that β-sheet (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). In species other thanE. coli and S. typhimurium, binding of CheR to chemoreceptors has yet to be investigated, but the sequences of CheR β-subdomains and of receptor carboxyl termini co-vary from the sequences in E. coli in a way that implies a conserved interaction (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). For the methylesterase CheB, less is known about the interaction of enzyme with the receptor pentapeptide. As a first step in learning more, we investigated the location of the NWETF-binding site. Like CheR, CheB has an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain (see Fig. 1). The catalytic domains of the two enzymes are structurally and presumably evolutionarily related (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar). Both are doubly wound α/β structures with a topological insertion. In CheR, this insertion is the β-sheet subdomain that binds the receptor pentapeptide (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). For CheB, the insertion is a β-hairpin positioned on the surface of the catalytic domain in the same relation to the enzyme active site as the pentapeptide-binding β-subdomain of CheR (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar). Thus the β-hairpin was a candidate for the pentapeptide-binding site in CheB. We investigated the location of the pentapeptide-binding site by experiments that tested catalytic activity and pentapeptide binding of CheB fragments, protection of CheB from proteolysis by pentapeptide, and interference with NWETF-CheB interaction by short CheB peptides. DISCUSSIONIn this study we used a combination of experimental approaches to locate the site on CheB at which this two-component response regulator and methylesterase/deamidase binds the pentapeptide present at the carboxyl-terminal of chemoreceptors. The results of these different approaches provided a consistent pattern that located the pentapeptide-binding site at the juncture between the carboxyl-terminal end of α-5, the final piece of secondary structure in the regulatory domain, and the linker that connects the regulatory and catalytic domains. This location is consistent with the lack of detectable interaction between pentapeptide and the catalytic domain of CheB, assessed by functional or binding assays. The location explains the binding of a CheB fragment containing α-5, linker and catalytic domain, the lack of binding by a related fragment containing only the linker and catalytic domain, the protection by free NWETF pentapeptide of a specific tryptic site at the boundary of α-5 and linker, and the interference with pentapeptide-CheB binding by an 11-residue segment of CheB that includes the boundary between α-5 and the linker.The experimentally determined location of the pentapeptide-binding site in CheB could not have been predicted by analogy with the structurally related methyltransferase CheR. In CheR, the receptor-binding site is provided by the β-subdomain, a topological insertion in the catalytic domain. In CheB, the analogous structural unit is a β-hairpin insertion in the catalytic domain (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). If the receptor-binding site were analogously placed on CheR and CheB, then the pentapeptide-binding site in CheB would be on the β-hairpin of the catalytic domain, not at the regulatory domain-linker boundary. Yet none of our experiments implicated the catalytic domain of CheB in receptor binding, but instead all the evidence pointed toward the carboxyl end of the regulatory domain.A difference in location of the receptor-binding site on CheR and CheB parallels other differences between interactions of the two modification enzymes with the receptor pentapeptide. In work to be reported elsewhere, we found that binding of CheB to the NWETF receptor sequence is substantially weaker than binding of CheR and that the interaction is likely to enhance enzyme action through effects on catalysis rather than enzyme recruitment. 2A. Barnakov, L. Barnakova, and G. L. Hazelbauer, manuscript in preparation. It is plausible that a binding site affecting catalysis would involve α-5 and the linker, because these parts of CheB participate in the interface between the regulatory and catalytic domain (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar, 43Hughes C.A. Mandell J.G. Anand G.S. Stock A.M. Komives E.A. J. Mol. Biol. 2001; 307: 967-976Crossref PubMed Scopus (53) Google Scholar), and interaction of those two domains is likely to limit enzyme action (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). Models of how the regulatory domain controls the activity of the catalytic domain suggest that in the low activity state access of substrate to the catalytic domain is restricted by the position of the regulatory domain (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar, 42Anand G.S. Goudreau P.N. Lewis J.K. Stock A.M. Protein Sci. 2000; 9: 898-906Crossref PubMed Scopus (16) Google Scholar). Yet phosphorylation of the regulatory domain has only subtle effects on the interface between the domains (43Hughes C.A. Mandell J.G. Anand G.S. Stock A.M. Komives E.A. J. Mol. Biol. 2001; 307: 967-976Crossref PubMed Scopus (53) Google Scholar), implying that phosphorylation-mediated enhancement of CheB action is not simply an extensive exposure of an otherwise occluded surface of the catalytic domain. Because pentapeptide-mediated enhancement of CheB action is independent of and additive with enhancement by regulatory domain phosphorylation (26Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar), it could be that pentapeptide binding near the hinge between the two domains creates a separation of the domains or affects the catalytic site directly in a way not caused by phosphorylation. There is no evidence that pentapeptide binding affects CheR catalysis. Instead all observations about enhancement of CheR action by pentapeptide can be explained by considering NWETF a high affinity docking site that recruits the methyltransferase to the region of its substrate methyl-accepting residues. Thus the difference in placement of the receptor-interaction sites on the two enzymes of adaptational modification parallels a difference in functional effects.The impressive sensitivity of the chemotactic system to very small changes in concentration of attractants indicates that signaling includes a means of creating significant gain, an amplification of small differences in receptor occupancy to produce detectable responses (44Mesibov R. Adler J. J. Bacteriol. 1972; 112: 315-326Crossref PubMed Google Scholar, 45Jasuja R. Keyoung J. Reid G.P. Trentham D.R. Khan S. Biophys. J. 1999; 76: 1706-1719Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The mechanism of this amplification is a subject of active research. A recent study implicates the enzymes of adaptational modification as crucial contributors to excitatory gain (46Kim C. Jackson M. Lux R. Khan S. J. Mol. Biol. 2001; 307: 119-135Crossref PubMed Scopus (46) Google Scholar). The authors of this study suggest that response sensitivity could be controlled by differential binding of the modification enzymes to distinct conformations of the chemoreceptors (46Kim C. Jackson M. Lux R. Khan S. J. Mol. Biol. 2001; 307: 119-135Crossref PubMed Scopus (46) Google Scholar). Knowledge of the sites of receptor interaction on CheB, as well as CheR, sets the stage for detailed investigation of this suggestion. In this study we used a combination of experimental approaches to locate the site on CheB at which this two-component response regulator and methylesterase/deamidase binds the pentapeptide present at the carboxyl-terminal of chemoreceptors. The results of these different approaches provided a consistent pattern that located the pentapeptide-binding site at the juncture between the carboxyl-terminal end of α-5, the final piece of secondary structure in the regulatory domain, and the linker that connects the regulatory and catalytic domains. This location is consistent with the lack of detectable interaction between pentapeptide and the catalytic domain of CheB, assessed by functional or binding assays. The location explains the binding of a CheB fragment containing α-5, linker and catalytic domain, the lack of binding by a related fragment containing only the linker and catalytic domain, the protection by free NWETF pentapeptide of a specific tryptic site at the boundary of α-5 and linker, and the interference with pentapeptide-CheB binding by an 11-residue segment of CheB that includes the boundary between α-5 and the linker. The experimentally determined location of the pentapeptide-binding site in CheB could not have been predicted by analogy with the structurally related methyltransferase CheR. In CheR, the receptor-binding site is provided by the β-subdomain, a topological insertion in the catalytic domain. In CheB, the analogous structural unit is a β-hairpin insertion in the catalytic domain (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). If the receptor-binding site were analogously placed on CheR and CheB, then the pentapeptide-binding site in CheB would be on the β-hairpin of the catalytic domain, not at the regulatory domain-linker boundary. Yet none of our experiments implicated the catalytic domain of CheB in receptor binding, but instead all the evidence pointed toward the carboxyl end of the regulatory domain. A difference in location of the receptor-binding site on CheR and CheB parallels other differences between interactions of the two modification enzymes with the receptor pentapeptide. In work to be reported elsewhere, we found that binding of CheB to the NWETF receptor sequence is substantially weaker than binding of CheR and that the interaction is likely to enhance enzyme action through effects on catalysis rather than enzyme recruitment. 2A. Barnakov, L. Barnakova, and G. L. Hazelbauer, manuscript in preparation. It is plausible that a binding site affecting catalysis would involve α-5 and the linker, because these parts of CheB participate in the interface between the regulatory and catalytic domain (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar, 43Hughes C.A. Mandell J.G. Anand G.S. Stock A.M. Komives E.A. J. Mol. Biol. 2001; 307: 967-976Crossref PubMed Scopus (53) Google Scholar), and interaction of those two domains is likely to limit enzyme action (36Djordjevic S. Stock A.M. Nat. Struct. Biol. 1998; 5: 446-450Crossref PubMed Scopus (77) Google Scholar). Models of how the regulatory domain controls the activity of the catalytic domain suggest that in the low activity state access of substrate to the catalytic domain is restricted by the position of the regulatory domain (37Djordjevic S. Goudreau P.N. Xu Q. Stock A.M. West A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1381-1386Crossref PubMed Scopus (156) Google Scholar, 42Anand G.S. Goudreau P.N. Lewis J.K. Stock A.M. Protein Sci. 2000; 9: 898-906Crossref PubMed Scopus (16) Google Scholar). Yet phosphorylation of the regulatory domain has only subtle effects on the interface between the domains (43Hughes C.A. Mandell J.G. Anand G.S. Stock A.M. Komives E.A. J. Mol. Biol. 2001; 307: 967-976Crossref PubMed Scopus (53) Google Scholar), implying that phosphorylation-mediated enhancement of CheB action is not simply an extensive exposure of an otherwise occluded surface of the catalytic domain. Because pentapeptide-mediated enhancement of CheB action is independent of and additive with enhancement by regulatory domain phosphorylation (26Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar), it could be that pentapeptide binding near the hinge between the two domains creates a separation of the domains or affects the catalytic site directly in a way not caused by phosphorylation. There is no evidence that pentapeptide binding affects CheR catalysis. Instead all observations about enhancement of CheR action by pentapeptide can be explained by considering NWETF a high affinity docking site that recruits the methyltransferase to the region of its substrate methyl-accepting residues. Thus the difference in placement of the receptor-interaction sites on the two enzymes of adaptational modification parallels a difference in functional effects. The impressive sensitivity of the chemotactic system to very small changes in concentration of attractants indicates that signaling includes a means of creating significant gain, an amplification of small differences in receptor occupancy to produce detectable responses (44Mesibov R. Adler J. J. Bacteriol. 1972; 112: 315-326Crossref PubMed Google Scholar, 45Jasuja R. Keyoung J. Reid G.P. Trentham D.R. Khan S. Biophys. J. 1999; 76: 1706-1719Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The mechanism of this amplification is a subject of active research. A recent study implicates the enzymes of adaptational modification as crucial contributors to excitatory gain (46Kim C. Jackson M. Lux R. Khan S. J. Mol. Biol. 2001; 307: 119-135Crossref PubMed Scopus (46) Google Scholar). The authors of this study suggest that response sensitivity could be controlled by differential binding of the modification enzymes to distinct conformations of the chemoreceptors (46Kim C. Jackson M. Lux R. Khan S. J. Mol. Biol. 2001; 307: 119-135Crossref PubMed Scopus (46) Google Scholar). Knowledge of the sites of receptor interaction on CheB, as well as CheR, sets the stage for detailed investigation of this suggestion. We thank A. Lilly for construction of plasmids, F. W. Dahlquist for pCW/CheB, J. S. Parkinson for RP3098, W. Siems for mass spectroscopy, and G. Munske for synthesis of peptides and for analyses by amino-terminal sequences. This work was begun while the authors were at Washington State University, Pullman, WA 99164-4660.