Uncoupling of Ligand-binding Affinity of the Bacterial Serine Chemoreceptor from Methylation- and Temperature-modulated Signaling States

Abstract

The Escherichia coli chemoreceptor Tsr mediates tactic responses to serine, repellents, and changes in temperature. We have previously shown that the serine-sensing ability of Tsr-T156C, which has a unique cysteine in place of threonine at residue 156, is specifically inactivated by thiol-modifying reagents and that l-serine protects the receptor from modification. In this study, we demonstrated the correlation between protective effects of various attractants and their potencies to elicit attractant responses. This indirect binding assay was used to monitor the affinity of the receptor for l-serine under various conditions. It has been demonstrated by in vitro assays that the ligand-binding affinities of Tsr and the related chemoreceptor Tar are unaffected by changes in the methylation state of the receptor. Using the serine protection assay, we re-examined this issue both in vitro and in vivo. The methylation levels of Tsr-T156C did not affect its ligand-binding affinity. We also showed both in vitro and in vivo that the ligand-binding affinity was unaffected by temperature. These results suggest that the structure of the periplasmic domain of the receptor is uncoupled from the signaling states of the cytoplasmic domain. This ligand-binding assay system should be applicable to other receptors. The Escherichia coli chemoreceptor Tsr mediates tactic responses to serine, repellents, and changes in temperature. We have previously shown that the serine-sensing ability of Tsr-T156C, which has a unique cysteine in place of threonine at residue 156, is specifically inactivated by thiol-modifying reagents and that l-serine protects the receptor from modification. In this study, we demonstrated the correlation between protective effects of various attractants and their potencies to elicit attractant responses. This indirect binding assay was used to monitor the affinity of the receptor for l-serine under various conditions. It has been demonstrated by in vitro assays that the ligand-binding affinities of Tsr and the related chemoreceptor Tar are unaffected by changes in the methylation state of the receptor. Using the serine protection assay, we re-examined this issue both in vitro and in vivo. The methylation levels of Tsr-T156C did not affect its ligand-binding affinity. We also showed both in vitro and in vivo that the ligand-binding affinity was unaffected by temperature. These results suggest that the structure of the periplasmic domain of the receptor is uncoupled from the signaling states of the cytoplasmic domain. This ligand-binding assay system should be applicable to other receptors. In many sensory systems, desensitization or adaptation to a persisting stimulus plays a crucial role in highly sensitive detection of stimuli over a comprehensive range. Extracellular signals (e.g. binding of ligands) are received and transduced into intracellular signals (e.g. activation or inactivation of kinases) by cell surface receptors. Binding of a ligand to a receptor extracellular domain induces some changes in structure of a receptor intracellular domain and, hence, in its activity. By contrast, upon adaptation, the signaling activities of the intracellular domains are often down-regulated by intracellular feedback regulatory systems (typically via covalent modification of the receptors). The effects of receptor down-regulation may be limited to the intracellular domain or may induce a global structural change in the receptor.Four closely related chemoreceptors of Escherichia coliserve as model systems for investigating both transmembrane signaling and down-regulation (for reviews, see Refs. 1Hazelbauer G.L. Yaghmai R. Burrows G.G. Baumgartner J.W. Dutton D.P. Morgan D.G. Armitage J.P. Lackie J.M. Biology of the Chemotactic Response. 46. Society for General Microbiology Symposium, Cambridge University Press, Cambridge, UK1990: 107-134Google Scholar, 2Bourret R.B. Borkovich K.A. Simon M.I. Annu. Rev. Biochem. 1991; 60: 401-441Google Scholar, 3Stock J.B. Lukat G.S. Stock A.M. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 109-136Google Scholar, 4Manson M.D. Adv. Microb. Physiol. 1992; 33: 277-346Google Scholar, 5Parkinson J.S. Cell. 1993; 73: 857-871Google Scholar, 6Stock J.B. Surette M.G. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1103-1129Google Scholar), Tsr (for serine), Tar (for aspartate and maltose), Trg (for ribose and galactose), and Tap (for dipeptide). These chemoreceptors function as homodimers regardless of ligand occupancy state (7Milligan D.L. Koshland Jr., D.E. J. Biol. Chem. 1988; 263: 6268-6275Google Scholar), and the homodimer forms a ternary complex with a homodimer of a cytoplasmic autokinase CheA and two molecules of an adaptor protein CheW (8Gegner J.A. Graham D.R. Roth A.F. Dahlquist F.W. Cell. 1992; 70: 975-982Google Scholar). Each receptor monomer consists of an N-terminal periplasmic ligand-binding domain, a C-terminal cytoplasmic signaling domain, and two membrane-spanning segments. Binding of a ligand to the interface between the two periplasmic domains triggers some structural change within the receptor dimer. This structural change leads to activation or inactivation of autophosphorylation of CheA and phosphotransfer from CheA to CheY. Phosphorylated CheY binds to the flagellar motor and induce clockwise (CW) 1The abbreviations used are: CW, clockwise; CCW, counterclockwise; Em, a methylated glutamic acid residue; NEM,N-ethylmaleimide; IPTG, isopropyl-1-thio-β-d-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TM1, the first transmembrane segment; TM2, the second transmembrane segment; Cmr, resistance to chloramphenicol; Tcr, resistance to tetracycline. 1The abbreviations used are: CW, clockwise; CCW, counterclockwise; Em, a methylated glutamic acid residue; NEM,N-ethylmaleimide; IPTG, isopropyl-1-thio-β-d-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TM1, the first transmembrane segment; TM2, the second transmembrane segment; Cmr, resistance to chloramphenicol; Tcr, resistance to tetracycline. rotation of the motor (causing tumbling of the cell), which otherwise rotates counterclockwise (CCW) (causing smooth swimming).Adaptation is achieved via methylation and demethylation of the chemoreceptors (9Springer M.S. Goy M.F. Adler J. Nature. 1979; 280: 279-280Google Scholar). Each chemoreceptor contains four to six glutamic acid residues that are reversibly methylated. Methyltransferase CheR catalyzes transfer of a methyl group fromS-adenosylmethionine to a glutamic acid side chain, and methylesterase CheB hydrolyzes the methyl ester bond of a methylated glutamate residue. The latter enzyme also serves as deamidase, converting specific glutamine residues of the nascent chemoreceptors to methylatable glutamic acid residues (10Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Google Scholar).Recent in vitro studies (11Borkovich K.A. Alex L.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6756-6760Google Scholar, 12Dunten P. Koshland Jr., D.E. J. Biol. Chem. 1991; 266: 1491-1496Google Scholar, 13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar) suggest that methylation of a receptor modulates its signaling state but has little effect on its ligand-binding affinity. This means that the structure of the periplasmic ligand-binding domain is uncoupled from the signaling state of the cytoplasmic domain under some conditions. However, contradictory results have been reported for receptor-containing membranes (14Yonekawa H. Hayashi H. FEBS Lett. 1986; 198: 21-24Google Scholar). To explain the discrepancy, Lin et al. (13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar) suggested that association of chemoreceptors with cytoplasmic proteins, such as CheA and CheW, might influence their ligand-binding behavior. Therefore, the effects of methylation and demethylation on ligand-binding affinity of receptors must be investigated under conditions closer to their native setting.The four chemoreceptors are unique in that they function also as thermosensors (15Imae Y. Eisenbach M. Balaban M. Sensing and Response in Microorganisms. Elsevier Science Publishing Co., Inc., New York1985: 73-81Google Scholar, 16Nara T. Lee L. Imae Y. J. Bacteriol. 1991; 173: 1120-1124Google Scholar). Tsr, Tar, and Trg are warm receptors that produce CCW signals upon a temperature increase and CW signals upon a temperature decrease, whereas Tap is a receptor with signaling T. Lee L. Imae Y. J. Bacteriol. 1991; 173: 1120-1124Google Scholar). should affect the signaling states of the but this be a change in temperature a global change in receptor structure or some change within the cytoplasmic signaling this study, we investigated the effects of and temperature on the ligand-binding affinity of We have previously shown that a attractant l-serine protects Tsr-T156C, which has a unique cysteine residue at the ligand-binding from thiol-modifying reagents such as in a T. S. M. Imae Y. J. Bacteriol. Scholar). This protective effect of l-serine should the affinity of Tsr-T156C for this assay be used to monitor the ligand-binding affinity of the receptor under various conditions. In this we a correlation between the stimulus of various acid attractants by Tsr-T156C and their to from modification. Using this assay system both in vitro and in we that the ligand-binding affinity of Tsr is uncoupled from its signaling states by covalent modification and used modification to the ligand-binding affinity of Tsr-T156C under various conditions. an attractant binds reversibly to Tsr, to the cysteine residue. Therefore, the of to Tsr-T156C in the of the In this study, we not of the be for such as demonstrated for between an enzyme and its or M.F. J. Biol. Chem. Scholar). However, Tsr-T156C was with for a of in the or the of protection with the affinities of Tsr-T156C for Therefore, we affinities of Tsr for l-serine under various conditions by the protective effect on modification a of this assay we that the affinity of Tsr is uncoupled from its signaling states vivo and in This is with in vitro studies on Tar (11Borkovich K.A. Alex L.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6756-6760Google Scholar, 12Dunten P. Koshland Jr., D.E. J. Biol. Chem. 1991; 266: 1491-1496Google Scholar) and Tsr (13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar). These results suggest that transmembrane from the to the of the cell not in However, a (14Yonekawa H. Hayashi H. FEBS Lett. 1986; 198: 21-24Google Scholar) demonstrated that methylation of Tsr and Tar the ligand affinity of receptors in the The might be by the that the latter did not monitor ligand binding to the receptors or by in the of the receptors in the the cytoplasmic proteins, CheA and CheW, which a ternary complex with the might to of the ligand-binding affinity upon which be for the as has been (13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar). We this Tsr-T156C was in and some not be in ternary with CheA and we that the affinity of Tsr is also uncoupled from its signaling states in vivo and in a change in temperature in the between and may not a change in the receptor such as or a but may some effects limited to the cytoplasmic domain. These structural changes should be the of by the The of the cytoplasmic domain in by Tar has been suggested by a T. S. M. Imae Y. J. Biol. Chem. Scholar), which showed that the of not have ability and the and the methylated forms of Tar function as warm and In this Tsr The as a warm receptor and to ability upon methylation Imae Y. J. Bacteriol. 1979; Scholar, L. T. Imae Y. J. Bacteriol. 1988; Scholar). These between Tsr and Tar might be related to in the of methylation M.R. Dahlquist F.W. Cell. Scholar, M.S. Dahlquist F.W. J. Biol. Chem. 1991; 266: Scholar). In covalent modification in the cytoplasmic domain the of the a which is with the that the structure of the cytoplasmic domain is by temperature results into the of the receptor. the ligand-binding domain may be in its is suggested by G.G. D.L. J. J. Koshland Jr., D.E. 1991; Scholar, J. Koshland D.E. J. Biol. Chem. 1993; Scholar, D.L. G.G. J. Koshland Jr., D.E. J. Biol. 1993; Scholar). is a between the and of a periplasmic of studies suggest that the first transmembrane and and binding of the ligand some of the second transmembrane to Proc. Natl. Acad. Sci. U. S. A. Scholar, Koshland Jr., D.E. Biochemistry. 1994; 33: Scholar, M.R. G.L. Proc. Natl. Acad. U. S. A. Scholar, Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; Scholar). However, to the cytoplasmic domain. in vitro studies suggest that of the cytoplasmic domain of Tar plays a role D.G. Weis R.M. Biochemistry. 1992; Scholar, Scholar). the other the assays for between two receptors suggest that cytoplasmic domain receptor is for signaling M. Scholar, M.D. Scholar). Therefore, the signaling may between changes within a cytoplasmic domain, or In binding of an attractant by a change in the periplasmic domain triggers a inactivation of CheA Therefore, is to that the two signaling states and on and of the receptor are by a structural change of the cytoplasmic domain. covalent modification and temperature changes should structural changes in the cytoplasmic domain. However, under such the structure of the periplasmic domain structural between the two domains the In many sensory systems, desensitization or adaptation to a persisting stimulus plays a crucial role in highly sensitive detection of stimuli over a comprehensive range. Extracellular signals (e.g. binding of ligands) are received and transduced into intracellular signals (e.g. activation or inactivation of kinases) by cell surface receptors. Binding of a ligand to a receptor extracellular domain induces some changes in structure of a receptor intracellular domain and, hence, in its activity. By contrast, upon adaptation, the signaling activities of the intracellular domains are often down-regulated by intracellular feedback regulatory systems (typically via covalent modification of the receptors). The effects of receptor down-regulation may be limited to the intracellular domain or may induce a global structural change in the receptor. closely related chemoreceptors of Escherichia coliserve as model systems for investigating both transmembrane signaling and down-regulation (for reviews, see Refs. 1Hazelbauer G.L. Yaghmai R. Burrows G.G. Baumgartner J.W. Dutton D.P. Morgan D.G. Armitage J.P. Lackie J.M. Biology of the Chemotactic Response. 46. Society for General Microbiology Symposium, Cambridge University Press, Cambridge, UK1990: 107-134Google Scholar, 2Bourret R.B. Borkovich K.A. Simon M.I. Annu. Rev. Biochem. 1991; 60: 401-441Google Scholar, 3Stock J.B. Lukat G.S. Stock A.M. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 109-136Google Scholar, 4Manson M.D. Adv. Microb. Physiol. 1992; 33: 277-346Google Scholar, 5Parkinson J.S. Cell. 1993; 73: 857-871Google Scholar, 6Stock J.B. Surette M.G. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1103-1129Google Scholar), Tsr (for serine), Tar (for aspartate and maltose), Trg (for ribose and galactose), and Tap (for dipeptide). These chemoreceptors function as homodimers regardless of ligand occupancy state (7Milligan D.L. Koshland Jr., D.E. J. Biol. Chem. 1988; 263: 6268-6275Google Scholar), and the homodimer forms a ternary complex with a homodimer of a cytoplasmic autokinase CheA and two molecules of an adaptor protein CheW (8Gegner J.A. Graham D.R. Roth A.F. Dahlquist F.W. Cell. 1992; 70: 975-982Google Scholar). Each receptor monomer consists of an N-terminal periplasmic ligand-binding domain, a C-terminal cytoplasmic signaling domain, and two membrane-spanning segments. Binding of a ligand to the interface between the two periplasmic domains triggers some structural change within the receptor dimer. This structural change leads to activation or inactivation of autophosphorylation of CheA and phosphotransfer from CheA to CheY. Phosphorylated CheY binds to the flagellar motor and induce clockwise (CW) 1The abbreviations used are: CW, clockwise; CCW, counterclockwise; Em, a methylated glutamic acid residue; NEM,N-ethylmaleimide; IPTG, isopropyl-1-thio-β-d-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TM1, the first transmembrane segment; TM2, the second transmembrane segment; Cmr, resistance to chloramphenicol; Tcr, resistance to tetracycline. 1The abbreviations used are: CW, clockwise; CCW, counterclockwise; Em, a methylated glutamic acid residue; NEM,N-ethylmaleimide; IPTG, isopropyl-1-thio-β-d-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TM1, the first transmembrane segment; TM2, the second transmembrane segment; Cmr, resistance to chloramphenicol; Tcr, resistance to tetracycline. rotation of the motor (causing tumbling of the cell), which otherwise rotates counterclockwise (CCW) (causing smooth is achieved via methylation and demethylation of the chemoreceptors (9Springer M.S. Goy M.F. Adler J. Nature. 1979; 280: 279-280Google Scholar). Each chemoreceptor contains four to six glutamic acid residues that are reversibly methylated. Methyltransferase CheR catalyzes transfer of a methyl group fromS-adenosylmethionine to a glutamic acid side chain, and methylesterase CheB hydrolyzes the methyl ester bond of a methylated glutamate residue. The latter enzyme also serves as deamidase, converting specific glutamine residues of the nascent chemoreceptors to methylatable glutamic acid residues (10Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Google Scholar). in vitro studies (11Borkovich K.A. Alex L.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6756-6760Google Scholar, 12Dunten P. Koshland Jr., D.E. J. Biol. Chem. 1991; 266: 1491-1496Google Scholar, 13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar) suggest that methylation of a receptor modulates its signaling state but has little effect on its ligand-binding affinity. This means that the structure of the periplasmic ligand-binding domain is uncoupled from the signaling state of the cytoplasmic domain under some conditions. However, contradictory results have been reported for receptor-containing membranes (14Yonekawa H. Hayashi H. FEBS Lett. 1986; 198: 21-24Google Scholar). To explain the discrepancy, Lin et al. (13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar) suggested that association of chemoreceptors with cytoplasmic proteins, such as CheA and CheW, might influence their ligand-binding behavior. Therefore, the effects of methylation and demethylation on ligand-binding affinity of receptors must be investigated under conditions closer to their native The four chemoreceptors are unique in that they function also as thermosensors (15Imae Y. Eisenbach M. Balaban M. Sensing and Response in Microorganisms. Elsevier Science Publishing Co., Inc., New York1985: 73-81Google Scholar, 16Nara T. Lee L. Imae Y. J. Bacteriol. 1991; 173: 1120-1124Google Scholar). Tsr, Tar, and Trg are warm receptors that produce CCW signals upon a temperature increase and CW signals upon a temperature decrease, whereas Tap is a receptor with signaling T. Lee L. Imae Y. J. Bacteriol. 1991; 173: 1120-1124Google Scholar). should affect the signaling states of the but this be a change in temperature a global change in receptor structure or some change within the cytoplasmic signaling In this study, we investigated the effects of and temperature on the ligand-binding affinity of We have previously shown that a attractant l-serine protects Tsr-T156C, which has a unique cysteine residue at the ligand-binding from thiol-modifying reagents such as in a T. S. M. Imae Y. J. Bacteriol. Scholar). This protective effect of l-serine should the affinity of Tsr-T156C for this assay be used to monitor the ligand-binding affinity of the receptor under various conditions. In this we a correlation between the stimulus of various acid attractants by Tsr-T156C and their to from modification. Using this assay system both in vitro and in we that the ligand-binding affinity of Tsr is uncoupled from its signaling states by covalent modification and temperature. used modification to the ligand-binding affinity of Tsr-T156C under various conditions. an attractant binds reversibly to Tsr, to the cysteine residue. Therefore, the of to Tsr-T156C in the of the In this study, we not of the be for such as demonstrated for between an enzyme and its or M.F. J. Biol. Chem. Scholar). However, Tsr-T156C was with for a of in the or the of protection with the affinities of Tsr-T156C for Therefore, we affinities of Tsr for l-serine under various conditions by the protective effect on modification a of this assay we that the affinity of Tsr is uncoupled from its signaling states vivo and in This is with in vitro studies on Tar (11Borkovich K.A. Alex L.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6756-6760Google Scholar, 12Dunten P. Koshland Jr., D.E. J. Biol. Chem. 1991; 266: 1491-1496Google Scholar) and Tsr (13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar). These results suggest that transmembrane from the to the of the cell not in However, a (14Yonekawa H. Hayashi H. FEBS Lett. 1986; 198: 21-24Google Scholar) demonstrated that methylation of Tsr and Tar the ligand affinity of receptors in the The might be by the that the latter did not monitor ligand binding to the receptors or by in the of the receptors in the the cytoplasmic proteins, CheA and CheW, which a ternary complex with the might to of the ligand-binding affinity upon which be for the as has been (13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar). We this Tsr-T156C was in and some not be in ternary with CheA and we that the affinity of Tsr is also uncoupled from its signaling states in vivo and in a change in temperature in the between and may not a change in the receptor such as or a but may some effects limited to the cytoplasmic domain. These structural changes should be the of by the The of the cytoplasmic domain in by Tar has been suggested by a T. S. M. Imae Y. J. Biol. Chem. Scholar), which showed that the of not have ability and the and the methylated forms of Tar function as warm and In this Tsr The as a warm receptor and to ability upon methylation Imae Y. J. Bacteriol. 1979; Scholar, L. T. Imae Y. J. Bacteriol. 1988; Scholar). These between Tsr and Tar might be related to in the of methylation M.R. Dahlquist F.W. Cell. Scholar, M.S. Dahlquist F.W. J. Biol. Chem. 1991; 266: Scholar). In covalent modification in the cytoplasmic domain the of the a which is with the that the structure of the cytoplasmic domain is by temperature results into the of the receptor. the ligand-binding domain may be in its is suggested by G.G. D.L. J. J. Koshland Jr., D.E. 1991; Scholar, J. Koshland D.E. J. Biol. Chem. 1993; Scholar, D.L. G.G. J. Koshland Jr., D.E. J. Biol. 1993; Scholar). is a between the and of a periplasmic of studies suggest that the first transmembrane and and binding of the ligand some of the second transmembrane to Proc. Natl. Acad. Sci. U. S. A. Scholar, Koshland Jr., D.E. Biochemistry. 1994; 33: Scholar, M.R. G.L. Proc. Natl. Acad. U. S. A. Scholar, Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; Scholar). However, to the cytoplasmic domain. in vitro studies suggest that of the cytoplasmic domain of Tar plays a role D.G. Weis R.M. Biochemistry. 1992; Scholar, Scholar). the other the assays for between two receptors suggest that cytoplasmic domain receptor is for signaling M. Scholar, M.D. Scholar). Therefore, the signaling may between changes within a cytoplasmic domain, or In binding of an attractant by a change in the periplasmic domain triggers a inactivation of CheA Therefore, is to that the two signaling states and on and of the receptor are by a structural change of the cytoplasmic domain. covalent modification and temperature changes should structural changes in the cytoplasmic domain. However, under such the structure of the periplasmic domain structural between the two domains the We used modification to the ligand-binding affinity of Tsr-T156C under various conditions. an attractant binds reversibly to Tsr, to the cysteine residue. Therefore, the of to Tsr-T156C in the of the In this study, we not of the be for such as demonstrated for between an enzyme and its or M.F. J. Biol. Chem. Scholar). However, Tsr-T156C was with for a of in the or the of protection with the affinities of Tsr-T156C for Therefore, we affinities of Tsr for l-serine under various conditions by the protective effect on modification a of Using this assay we that the affinity of Tsr is uncoupled from its signaling states vivo and in This is with in vitro studies on Tar (11Borkovich K.A. Alex L.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6756-6760Google Scholar, 12Dunten P. Koshland Jr., D.E. J. Biol. Chem. 1991; 266: 1491-1496Google Scholar) and Tsr (13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar). These results suggest that transmembrane from the to the of the cell not in However, a (14Yonekawa H. Hayashi H. FEBS Lett. 1986; 198: 21-24Google Scholar) demonstrated that methylation of Tsr and Tar the ligand affinity of receptors in the The might be by the that the latter did not monitor ligand binding to the receptors or by in the of the receptors in the the cytoplasmic proteins, CheA and CheW, which a ternary complex with the might to of the ligand-binding affinity upon which be for the as has been (13Lin L.-N. Li J. Brandts J.F. Weis R.M. Biochemistry. 1994; 33: 6564-6570Google Scholar). We this Tsr-T156C was in and some not be in ternary with CheA and we that the affinity of Tsr is also uncoupled from its signaling states in vivo and in a change in temperature in the between and may not a change in the receptor such as or a but may some effects limited to the cytoplasmic domain. These structural changes should be the of by the The of the cytoplasmic domain in by Tar has been suggested by a T. S. M. Imae Y. J. Biol. Chem. Scholar), which showed that the of not have ability and the and the methylated forms of Tar function as warm and In this Tsr The as a warm receptor and to ability upon methylation Imae Y. J. Bacteriol. 1979; Scholar, L. T. Imae Y. J. Bacteriol. 1988; Scholar). These between Tsr and Tar might be related to in the of methylation M.R. Dahlquist F.W. Cell. Scholar, M.S. Dahlquist F.W. J. Biol. Chem. 1991; 266: Scholar). In covalent modification in the cytoplasmic domain the of the a which is with the that the structure of the cytoplasmic domain is by temperature results into the of the receptor. the ligand-binding domain may be in its is suggested by G.G. D.L. J. J. Koshland Jr., D.E. 1991; Scholar, J. Koshland D.E. J. Biol. Chem. 1993; Scholar, D.L. G.G. J. Koshland Jr., D.E. J. Biol. 1993; Scholar). is a between the and of a periplasmic of studies suggest that the first transmembrane and and binding of the ligand some of the second transmembrane to Proc. Natl. Acad. Sci. U. S. A. Scholar, Koshland Jr., D.E. Biochemistry. 1994; 33: Scholar, M.R. G.L. Proc. Natl. Acad. U. S. A. Scholar, Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; Scholar). However, to the cytoplasmic domain. in vitro studies suggest that of the cytoplasmic domain of Tar plays a role D.G. Weis R.M. Biochemistry. 1992; Scholar, Scholar). the other the assays for between two receptors suggest that cytoplasmic domain receptor is for signaling M. Scholar, M.D. Scholar). Therefore, the signaling may between changes within a cytoplasmic domain, or In binding of an attractant by a change in the periplasmic domain triggers a inactivation of CheA Therefore, is to that the two signaling states and on and of the receptor are by a structural change of the cytoplasmic domain. covalent modification and temperature changes should structural changes in the cytoplasmic domain. However, under such the structure of the periplasmic domain structural between the two domains the We M. Simon for and S. and for in of We J. S. for the