Allosteric Enhancement of Adaptational Demethylation by a Carboxyl-terminal Sequence on Chemoreceptors
2002; Elsevier BV; Volume: 277; Issue: 44 Linguagem: Inglês
10.1074/jbc.m206245200
ISSN1083-351X
AutoresAlexander N. Barnakov, Ludmila A. Barnakova, Gerald L. Hazelbauer,
Tópico(s)Pharmacological Receptor Mechanisms and Effects
ResumoSensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors. Specific glutamyl residues are methylated and demethylated in reactions catalyzed by methyltransferase CheR and methylesterase CheB. In Escherichia coli and Salmonella enterica serovar typhimurium, efficient adaptational modification by either enzyme is dependent on a conserved pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from the sites of modification. For CheR-catalyzed methylation, previous work demonstrated that this sequence acts as a high affinity docking site, enhancing methylation by increasing enzyme concentration near methyl-accepting glutamates. We investigated pentapeptide-mediated enhancement of CheB-catalyzed demethylation and found it occurred by a distinctly different mechanism. Assays of binding between CheB and the pentapeptide sequence showed that it was too weak to have a significant effect on local enzyme concentration. Kinetic analyses revealed that interaction of the sequence and the methylesterase enhanced the rate constant of demethylation not the Michaelis constant. This allosteric activation occurred if the sequence was attached to chemoreceptor, but hardly at all if it was present as an isolated peptide. In addition, free peptide inhibited demethylation of the native receptor carrying the pentapeptide sequence at its carboxyl terminus. These observations imply that the allosteric change is transmitted through the protein substrate, not the enzyme. Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors. Specific glutamyl residues are methylated and demethylated in reactions catalyzed by methyltransferase CheR and methylesterase CheB. In Escherichia coli and Salmonella enterica serovar typhimurium, efficient adaptational modification by either enzyme is dependent on a conserved pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from the sites of modification. For CheR-catalyzed methylation, previous work demonstrated that this sequence acts as a high affinity docking site, enhancing methylation by increasing enzyme concentration near methyl-accepting glutamates. We investigated pentapeptide-mediated enhancement of CheB-catalyzed demethylation and found it occurred by a distinctly different mechanism. Assays of binding between CheB and the pentapeptide sequence showed that it was too weak to have a significant effect on local enzyme concentration. Kinetic analyses revealed that interaction of the sequence and the methylesterase enhanced the rate constant of demethylation not the Michaelis constant. This allosteric activation occurred if the sequence was attached to chemoreceptor, but hardly at all if it was present as an isolated peptide. In addition, free peptide inhibited demethylation of the native receptor carrying the pentapeptide sequence at its carboxyl terminus. These observations imply that the allosteric change is transmitted through the protein substrate, not the enzyme. Sensory systems are designed to adapt to persistent stimulation. In bacterial chemotaxis, adaptation is accomplished through covalent modification of transmembrane chemoreceptors (1Springer M.S. Goy M.F. Adler J. Nature. 1979; 280: 279-284Crossref PubMed Scopus (246) Google Scholar). Specific glutamyl residues in the chemoreceptor cytoplasmic domain (2Kehry 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 methylated by methyltransferase CheR (3Springer W.R. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 533-537Crossref PubMed Scopus (187) Google Scholar) to form carboxyl methyl esters. These esters can be hydrolyzed by methylesterase CheB (4Stock 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 coli andSalmonella enterica serovar typhimurium (see Refs. 5Falke 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 (419) Google Scholar, 6Hazelbauer G.L. Adelman G. Smith B.H. Encyclopedia of Neuroscience. Elsevier, Amsterdam1999: 181-183Google Scholar, 7Falke J.J. Hazelbauer G.L. Trends Biochem. Sci. 2001; 26: 257-265Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar for reviews), chemoreceptors have four to six methyl-accepting glutamyl residues, four of which are at conserved positions (2Kehry 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. Wang J.Y. Koshland Jr., D.E. J. Biol. Chem. 1986; 261: 10814-10820Abstract 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) (Fig.1). Control of receptor methylation and demethylation determines steady state cellular behavior, the ability of the chemosensory system to provide exact adaptation over a wide dynamic range, and the process of molecular memory. Thus the means by which receptor modification is modulated are of great interest.Chemoreceptors form complexes with the chemotaxis-specific histidine kinase CheA, enhancing an otherwise low rate of autophosphorylation (12Borkovich K.A. Kaplan N. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1208-1212Crossref PubMed Scopus (236) Google Scholar) and providing phosphoryl groups for transfer to two response regulators, CheY and CheB (13Hess 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 and determines the direction of rotation (14Welch 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 its regulatory domain, activating its catalytic domain (15Lupas A. Stock J. J. Biol. Chem. 1989; 264: 17337-17342Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). Binding of chemoattractant to receptor reduces kinase activity (12Borkovich K.A. Kaplan N. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1208-1212Crossref PubMed Scopus (236) Google Scholar) and thus cellular levels of phospho-CheY, resulting in a change in rotational bias and thus an altered pattern of swimming (16Barak R. Eisenbach M. Biochemistry. 1992; 31: 1821-1826Crossref PubMed Scopus (125) Google Scholar). However, these changes are short-lived because an increase in ligand occupancy also initiates the feedback loop of adaptation. Stimulated receptors accumulate methyl groups through a combination of a reduced cellular level of phospho-CheB and occupancy specific activation of methyl-accepting sites (17Borczuk A. Staub A. Stock J. Biochem. Biophys. Res. Commun. 1986; 141: 918-923Crossref PubMed Scopus (13) Google Scholar, 18Russell C.B. Stewart R.C. Dahlquist F.W. J. Bacteriol. 1989; 171: 3609-3618Crossref PubMed Google Scholar). Increased methylation creates a compensatory change in the receptor-kinase complex (19Borkovich K.A. Alex L.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6756-6760Crossref PubMed Scopus (157) Google Scholar) that restores CheA activity to its null, receptor-activated state even though the increased level of attractant persists.What determines the efficiency of adaptational methylation and demethylation? Sites of modification are spaced seven apart in the receptor sequence (2Kehry 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 chemoreceptor cytoplasmic domain (20Kim 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. Wang J.Y. Koshland Jr., D.E. J. Biol. Chem. 1986; 261: 10814-10820Abstract Full Text PDF PubMed Google Scholar, 21Shapiro 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 is at the chemoreceptor carboxyl terminus, separated from the sites of modification. In E. coli and Salmonella, a conserved sequence, asparagine-tryptophan-glutamate-threonine-phenylalanine (NWETF), at the carboxyl terminus of high abundance chemoreceptors (Fig. 1) interacts with the methyltransferase (22Wu J., Li, J., Li, G. Long D.G. Weis R.M. Biochemistry. 1996; 35: 4984-4993Crossref PubMed Scopus (153) Google Scholar) and the methylesterase (23Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar, 24Barnakov A.N. Barnakova L.A. Hazelbauer G.L. J. Biol. Chem. 2001; 276: 32984-32989Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Chemoreceptors lacking the pentapeptide naturally (the low-abundance receptors) or as the result of engineered truncations or mutations are inefficiently methylated, demethylated, and deamidated (23Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar,25Li J., Li, G. Weis R.M. Biochemistry. 1997; 36: 11851-11857Crossref PubMed Scopus (72) Google Scholar, 26Le Moual H. Quang T. Koshland Jr., D.E. Biochemistry. 1997; 36: 13441-13448Crossref PubMed Scopus (55) Google Scholar, 27Okumura H. Nishiyama S. Sasaki A. Homma M. Kawagishi I. J. Bacteriol. 1998; 180: 1862-1868Crossref PubMed Google Scholar, 28Feng 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 (28Feng X. Lilly A.A. Hazelbauer G.L. J. Bacteriol. 1999; 181: 3164-3171Crossref PubMed Google Scholar, 29Hazelbauer G.L. Engstrom P. Nature. 1980; 283: 98-100Crossref PubMed Scopus (45) Google Scholar, 30Yamamoto K. Macnab R.M. Imae Y. J. Bacteriol. 1990; 172: 383-388Crossref PubMed Google Scholar, 31Feng X. Baumgartner J.W. Hazelbauer G.L. J. Bacteriol. 1997; 179: 6714-6720Crossref PubMed Google Scholar, 32Weerasuriya 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 (28Feng X. Lilly A.A. Hazelbauer G.L. J. Bacteriol. 1999; 181: 3164-3171Crossref PubMed Google Scholar, 29Hazelbauer G.L. Engstrom P. Nature. 1980; 283: 98-100Crossref PubMed Scopus (45) Google Scholar, 31Feng X. Baumgartner J.W. Hazelbauer G.L. J. Bacteriol. 1997; 179: 6714-6720Crossref PubMed Google Scholar,32Weerasuriya S. Schneider B.M. Manson M.D. J. Bacteriol. 1998; 180: 914-920Crossref PubMed Google Scholar).How does the NWETF pentapeptide at the carboxyl terminus of a receptor enhance adaptational modification? For methyltransferase CheR, it provides a high affinity docking site that increases enzyme concentration near the methyl-accepting glutamates (22Wu J., Li, J., Li, G. Long D.G. Weis R.M. Biochemistry. 1996; 35: 4984-4993Crossref PubMed Scopus (153) Google Scholar). In the current work we investigated enhancement of CheB-catalyzed demethylation by the pentapeptide sequence. We found that it occurs through a distinctly different mechanism of allosteric activation.DISCUSSIONWe found that enhancement of CheB-catalyzed demethylation by the NWETF pentapeptide sequence at the carboxyl terminus of a chemoreceptor occurred in a way distinctly different from its enhancement of CheR-catalyzed methylation. For the methylation reaction, the sequence provides a high affinity docking site for the CheR methyltransferase and thus increases the effective concentration of the enzyme in the neighborhood of substrate methyl-accepting sites (22Wu J., Li, J., Li, G. Long D.G. Weis R.M. Biochemistry. 1996; 35: 4984-4993Crossref PubMed Scopus (153) Google Scholar). In contrast, interaction of CheB with receptor-borne pentapeptide enhanced demethylation allosterically. Consistent with an allosteric action, the sequence bound too weakly to the CheB methylesterase to cause a significant increase in local concentration of the enzyme.Different Mechanisms of EnhancementOf the two kinds of CheR-interaction sites on a chemoreceptor, the strongest binding is not at the sites of catalysis (the methyl-accepting glutamyl residues) but instead at the carboxyl-terminal NWETF sequence. CheR binds with the same dissociation constant of ∼2 μm to pentapeptide alone, to the isolated cytoplasmic domain of chemoreceptor Tsr carrying the pentapeptide sequence and three methyl-accepting sites, and to intact receptor carrying the sequence and from one to five methyl-accepting sites, but exhibits no detectable binding to isolated cytoplasmic domains containing methyl-accepting sites but missing the carboxyl-terminal sequence (22Wu J., Li, J., Li, G. Long D.G. Weis R.M. Biochemistry. 1996; 35: 4984-4993Crossref PubMed Scopus (153) Google Scholar). Differences in rates of methylation for Tar and TarΔpp imply that binding of CheR to its substrate sites is ∼20-fold weaker than binding to the pentapeptide sequence (26Le Moual H. Quang T. Koshland Jr., D.E. Biochemistry. 1997; 36: 13441-13448Crossref PubMed Scopus (55) Google Scholar). Taken together, these observations show that the sequence acts as a high affinity docking site for the methyltransferase, enhancing the methylation reaction by recruiting the enzyme to the vicinity of its substrate sites and thus increasing the probability of binding at the methyl-accepting glutamyl residues.Our data indicate that this is not the mechanism by which receptor-borne pentapeptide enhances CheB-catalyzed demethylation. Kinetic analysis of demethylation revealed that the NWETF sequence at the carboxyl terminus of a receptor substrate enhanced k cat not K m, and thus indicated that pentapeptide-enzyme interaction affected catalysis not binding. It is interesting to note that phosphorylation, which activates CheB independent of interaction with the pentapeptide (23Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar), also enhances k cat rather thanK m (39Anand G.S. Stock A.M. Biochemistry. 2002; 41: 6752-6760Crossref PubMed Scopus (22) Google Scholar). Enhancement of k catnot K m was consistent with the weak binding we observed for the enzyme and pentapeptide sequence, a binding insufficient to enhance enzyme action by increasing concentrations of CheB near substrate sites. As documented in Figs. 4 and 5, CheB bound to the free pentapeptide NWETF with a K d ∼ 150 μm, and the concentrations of free peptide necessary to inhibit phospho-CheB activity (Fig. 7) indicated that phosphorylation of the enzyme did not make the affinity substantially stronger. Investigation of CheB binding to the sequence at its natural location at the carboxyl terminus of a chemoreceptor indicated a similarly weak interaction. The short lifetime of phospho-CheB kept us from performing direct measurements of binding of phosphorylated enzyme to receptor-borne pentapeptide, but a strong binding for only this combination could not explain enhancement by the receptor-borne pentapeptide of demethylation catalyzed by CheB that is not phosphorylated (23Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar), nor the effect of the receptor-borne pentapeptide on k cat rather than K m. Estimates of the cellular content of chemoreceptors range from 2,500 to 10,000 (41Hazelbauer G.L. Engstrom P. Harayama S. J. Bacteriol. 1981; 145: 43-49Crossref PubMed Google Scholar, 42Clarke S. Koshland Jr., D.E. J. Biol. Chem. 1979; 254: 9695-9702Abstract Full Text PDF PubMed Google Scholar, 43Stock J.B. Koshland Jr., D.E. J. Biol. Chem. 1981; 256: 10826-10833Abstract Full Text PDF PubMed Google Scholar) and the content of CheB has been estimated at 2,000 (44Simms S.A. Stock A.M. Stock J.B. J. Biol. Chem. 1987; 262: 8537-8543Abstract Full Text PDF PubMed Google Scholar). These values translate to cellular concentrations between 4 and 16 μm for chemoreceptors and ∼3 μm CheB. At such concentrations, a CheB-pentapeptide complex with aK d of ∼150 μm, would involve only a small proportion of total cellular CheB and this complex would not create a significant increase in concentration of CheB in the neighborhood of its sites of enzymatic action.There is a disparity between the ∼150 μm K d of pentapeptide and CheB or phospho-CheB and theK m of ∼3 μm for demethylation by either CheB or phospho-CheB. We do not fully understand this disparity, but it can be understood if we assume that K mapproximates K d for enzyme and substrate methyl ester (see below for the conditions under which this assumption would be valid). In this case, the interaction of the enzyme and substrate site would be significantly stronger that the interaction of the enzyme and receptor-borne pentapeptide. This stronger binding, as reflected inK m, would not be a function of the presence or absence of the pentapeptide sequence on a receptor (this is what we observe), and allosteric enhancement of CheB-mediated catalysis would be the result of binding of pentapeptide to enzyme bound to or near the substrate site. Using K m to approximateK d is valid if the rate constant of dissociation for unreacted substrate from the enzyme-substrate complex is substantially higher than k cat and the catalytic mechanism does not involve a covalent intermediate. These issues are currently under investigation. No matter what the affinity of enzyme for its substrate sites, the weak affinity of enzyme for pentapeptide indicates that this interaction could not assist CheB in passing through a receptor array by the "molecular brachiation" process postulated for CheR (40Levin M.D. Shimizu T.S. Bray D. Biophys. J. 2002; 82: 1809-1817Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar).Kinetic parameters for phospho-CheB acting on NWETF-containing receptor, K m ∼ 2.8 μm andk cat ∼ 190 mmol of methanol s−1mol of CheB−1, are quite similar to the parameters for CheR, which are K m = 2.1 μm andk cat = 170 mmol s−1 mol of CheR−1 (44Simms S.A. Stock A.M. Stock J.B. J. Biol. Chem. 1987; 262: 8537-8543Abstract Full Text PDF PubMed Google Scholar). This is consistent with the dynamic equilibrium of adaptational modification, in which the population of unstimulated or adapted receptor is maintained at a constant level of methylation by a balance between rates of methylation and demethylation (45Kehry M.R. Doak T.G. Dahlquist F.W. J. Biol. Chem. 1984; 259: 11828-11835Abstract Full Text PDF PubMed Google Scholar).Allosteric Activation of DemethylationThe pentapeptide-binding site on CheB is separated from the active site (24Barnakov A.N. Barnakova L.A. Hazelbauer G.L. J. Biol. Chem. 2001; 276: 32984-32989Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and thus enhancement of catalysis through an effect on the enzyme would be an effect of binding at a site distance from the site of catalysis, i.e. an allosteric effect. However, the substrate itself is a protein, so it was possible that interaction of its carboxyl-terminal sequence with the methylesterase could allosterically alter the receptor to enhance demethylation. The data are consistent with this second alternative, that allosteric activation is principally through the receptor, not the enzyme. If binding of the NWETF sequence induced an allosteric activation in the methylesterase, then it should not be crucial whether this sequence were present in its natural location at the carboxyl terminus of a chemoreceptor or as a free pentapeptide. Fig. 6 documents that free pentapeptide provided only a few percent of the activation created by the receptor-borne sequence. If allosteric activation required that the pentapeptide sequence be coupled to the chemoreceptor, then free pentapeptide should inhibit demethylation of receptors carrying the carboxyl-terminal sequence. Fig. 7 documents that this is the case. Taken together, these two results imply that the vast majority of the enhancement of demethylation generated by interaction of the receptor-borne sequence and CheB is conveyed through a change in the receptor, not the enzyme. Thus the primary allosteric effect appears to be through the protein substrate. However, the issues require additional investigation, because we have not yet demonstrated directly induction of a conformation change in the receptor by interaction of CheB and receptor-borne pentapeptide. Such investigations are underway.Substrate-transmitted Allostery and Signal AmplificationChemotactic response in E. coli exhibits striking signal amplification (46Mesibov R. Ordal G.W. Adler J. J. Gen. Physiol. 1973; 62: 203-223Crossref PubMed Scopus (178) Google Scholar, 47Segall J.E. Block S.M. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8987-8991Crossref PubMed Scopus (377) Google Scholar, 48Kim C. Jackson M. Lux R. Khan S. J. Mol. Biol. 2001; 307: 119-135Crossref PubMed Scopus (46) Google Scholar, 49Sourjik V. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 123-127Crossref PubMed Scopus (393) Google Scholar). The mechanism of this amplification is not yet understood, but recent observations have implicated methylesterase CheB as a crucial participant (48Kim C. Jackson M. Lux R. Khan S. J. Mol. Biol. 2001; 307: 119-135Crossref PubMed Scopus (46) Google Scholar, 49Sourjik V. Berg H.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 123-127Crossref PubMed Scopus (393) Google Scholar). If CheB binding to receptor-borne, carboxyl-terminal pentapeptide induces substrate-transmitted allostery, this conformational change could be related to the role of CheB in signal amplification. Sensory systems are designed to adapt to persistent stimulation. In bacterial chemotaxis, adaptation is accomplished through covalent modification of transmembrane chemoreceptors (1Springer M.S. Goy M.F. Adler J. Nature. 1979; 280: 279-284Crossref PubMed Scopus (246) Google Scholar). Specific glutamyl residues in the chemoreceptor cytoplasmic domain (2Kehry 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 methylated by methyltransferase CheR (3Springer W.R. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 533-537Crossref PubMed Scopus (187) Google Scholar) to form carboxyl methyl esters. These esters can be hydrolyzed by methylesterase CheB (4Stock 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 coli andSalmonella enterica serovar typhimurium (see Refs. 5Falke 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 (419) Google Scholar, 6Hazelbauer G.L. Adelman G. Smith B.H. Encyclopedia of Neuroscience. Elsevier, Amsterdam1999: 181-183Google Scholar, 7Falke J.J. Hazelbauer G.L. Trends Biochem. Sci. 2001; 26: 257-265Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar for reviews), chemoreceptors have four to six methyl-accepting glutamyl residues, four of which are at conserved positions (2Kehry 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. Wang J.Y. Koshland Jr., D.E. J. Biol. Chem. 1986; 261: 10814-10820Abstract 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) (Fig.1). Control of receptor methylation and demethylation determines steady state cellular behavior, the ability of the chemosensory system to provide exact adaptation over a wide dynamic range, and the process of molecular memory. Thus the means by which receptor modification is modulated are of great interest. Chemoreceptors form complexes with the chemotaxis-specific histidine kinase CheA, enhancing an otherwise low rate of autophosphorylation (12Borkovich K.A. Kaplan N. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1208-1212Crossref PubMed Scopus (236) Google Scholar) and providing phosphoryl groups for transfer to two response regulators, CheY and CheB (13Hess 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 and determines the direction of rotation (14Welch 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 its regulatory domain, activating its catalytic domain (15Lupas A. Stock J. J. Biol. Chem. 1989; 264: 17337-17342Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). Binding of chemoattractant to receptor reduces kinase activity (12Borkovich K.A. Kaplan N. Hess J.F. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1208-1212Crossref PubMed Scopus (236) Google Scholar) and thus cellular levels of phospho-CheY, resulting in a change in rotational bias and thus an altered pattern of swimming (16Barak R. Eisenbach M. Biochemistry. 1992; 31: 1821-1826Crossref PubMed Scopus (125) Google Scholar). However, these changes are short-lived because an increase in ligand occupancy also initiates the feedback loop of adaptation. Stimulated receptors accumulate methyl groups through a combination of a reduced cellular level of phospho-CheB and occupancy specific activation of methyl-accepting sites (17Borczuk A. Staub A. Stock J. Biochem. Biophys. Res. Commun. 1986; 141: 918-923Crossref PubMed Scopus (13) Google Scholar, 18Russell C.B. Stewart R.C. Dahlquist F.W. J. Bacteriol. 1989; 171: 3609-3618Crossref PubMed Google Scholar). Increased methylation creates a compensatory change in the receptor-kinase complex (19Borkovich K.A. Alex L.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6756-6760Crossref PubMed Scopus (157) Google Scholar) that restores CheA activity to its null, receptor-activated state even though the increased level of attractant persists. What determines the efficiency of adaptational methylation and demethylation? Sites of modification are spaced seven apart in the receptor sequence (2Kehry 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 chemoreceptor cytoplasmic domain (20Kim 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. Wang J.Y. Koshland Jr., D.E. J. Biol. Chem. 1986; 261: 10814-10820Abstract Full Text PDF PubMed Google Scholar, 21Shapiro 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 is at the chemoreceptor carboxyl terminus, separated from the sites of modification. In E. coli and Salmonella, a conserved sequence, asparagine-tryptophan-glutamate-threonine-phenylalanine (NWETF), at the carboxyl terminus of high abundance chemoreceptors (Fig. 1) interacts with the methyltransferase (22Wu J., Li, J., Li, G. Long D.G. Weis R.M. Biochemistry. 1996; 35: 4984-4993Crossref PubMed Scopus (153) Google Scholar) and the methylesterase (23Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10667-10672Crossref PubMed Scopus (74) Google Scholar, 24Barnakov A.N. Barnakova L.A. Hazelbauer G.L. J. Biol. Chem. 2001; 276: 32984-32989Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Chemoreceptors lacking the pentapeptide naturally (the low-abundance receptors) or as the result of engineered truncations or mutations are inefficiently methylated, demethylated, and deamidated (23Barnakov A.N. Barnakova L.A. Hazelbauer G.L. Proc. Natl. Acad. Sci. U. S. 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