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Enzymatic Deamidation of Methyl-Accepting Chemotaxis

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Enzymatic Deamidation of Methyl-Accepting Chemotaxis Proc. NatL Acad. Sci. USA Vol. 80, pp. 3599-3603, June 1983 Biochemistry Enzymatic deamidation of methyl-accepting chemotaxis proteins in Escherichia coli catalyzed by the cheB gene product (methylation/CheB-dependent modification/sensory adaptation) MARILYN R. KEHRY*, MARTHA W. BONDtt, MICHAEL W. HUNKAPILLERt, AND FREDERICK W. DAHLQUIST* *Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403; and tDivision of Biology 156-29, California Institute of Technology, Pasadena, California 91125 Communicated by George Streisinger, March 11, 1983 ABSTRACT The methyl-accepting chemotaxis proteins (MCPs) methyl ester of glutamic acid (20-23). A protein methylesterase of Escherichia coli undergo reversible methylation that has been encoded by the cheB gene balances the methyltransferase ac- correlated with adaptation of cells to environmental stimuli. MCPI, tivity by removing methyl groups (24, 25). Each MCP has mul- the product of the tsr gene, accepts methyl groups at multiple sites tiple sites at which methylation and demethylation may occur that are located on two tryptic peptides, denoted KI and RI. A (26-29). For example, MCPI may accept a maximum of six second modification of the MCPs, which is not methylation, has methyl groups, and MCPII accepts at least four methyl groups been designated the CheB-dependent modification. A CheB-de- (30, 31). pendent modification occurs on methyl-accepting peptide KI and A second covalent modification of the MCPs also occurs at allows additional methyl groups to be incorporated into this pep- multiple sites and is dependent on the presence of the cheB tide. We have performed partial amino acid sequence analyses on radiolabeled peptides Ki and RI derived from MCPI and have gene (32, 33). The chemical nature of the modification, de- identified several methyl-accepting sites. We found that, in the noted the CheB-dependent modification, had not been iden- absence of CheB-dependent modification, a site in peptide Ki is tified previously, and its specific role in chemotaxis is unclear. unable to accept methyl groups. Correlation of this protein se- However, several properties suggest that CheB-dependent quence data with the nucleotide sequence of the tsr gene [Boyd, modification may be the enzymatic conversion of glutamine to A., Kendall, K. & Simon, M. I. (1983) Nature (London) 301, 623- glutamic acid residues (32). Each CheB modification of MCPs 626] suggests that CheB-dependent modification of MCPI is the results in the addition of one net negative charge to the protein enzymatic deamidation of glutamine to methyl-accepting glutamic and allows additional methyl groups to be accepted by the MCP acid. Possible roles for this deamidation in bacterial chemotaxis (31, 32). are discussed. We have previously shown (30) that two tryptic peptides ac- cept methyl groups in MCPI and MCPII. One of these, des- Escherichia coli swim in a random walk resulting from alter- ignated peptide K1, contains lysine and methionine; the other, nating clockwise (tumbling behavior) and counterclockwise designated peptide RI, contains arginine. In addition, peptide (straight swimming behavior) flagellar rotation. The bacteria K1 derived from MCPI contains at least one CheB-modifiable respond to changes in their environment by modulating the site. To identify the CheB-modifiable amino acid(s) and elu- probabilities of clockwise and counterclockwise rotations. Cells cidate the chemical nature of the CheB-dependent modifica- suppress their tumbling in response to attractants and increase tion, we have performed protein sequence determinations on their tumbling in the presence of repellents. Subsequently, the the radiolabeled K1 and RI peptides derived from MCPI. One cells undergo a process of adaptation, which entails a return to of the methylated positions observed in peptide K1 that is their prestimulus swimming pattern (1-3). methylated in protein obtained from a wild-type background These behavioral responses of E. coli to certain stimuli are remains completely unmethylated in protein synthesized in a mediated by a family of transmembrane proteins, the methyl- cheB mutant. In conjunction with the amino acid sequence of accepting chemotaxis proteins (MCPs) (4). At least three dif- MCPI deduced from the nucleotide sequence of the tsr gene ferent MCPs are essential in the sensory transduction process. (34), these data provide compelling evidence that CheB mod- MCPI, MCPII, and MCPIII, encoded by the tsr, tar, and trg ification is the enzymatic deamidation of glutamine to glutamic genes, respectively, mediate cellular responses to different acid. Possible functions for this deamidation in chemotaxis are subsets of attractants and repellents (5-8). A fourth gene, tap, discussed. seems to encode an additional MCP whose functional role in chemotaxis has not yet been defined (9-11). The behavioral re- MATERIALS AND METHODS sponse is initiated by interaction of the effector molecule or an Strains. E. coli strains and A-E. coli hybrid phage were as effector-receptor complex in the periplasm with the appropri- described (30, 31). All strains and phages were the generous ate MCP (12-16). gifts of J. S. Parkinson and M. Simon. MCPs undergo reversible methylations in the cell cyto- Radiolabeling of MCPs, Electrophoresis, Enzyme Diges- plasm, and changes in the level of methylation correlate with tions, HPLC Analysis, and Isolation of Peptides. These pro- the adaptation of cells to environmental stimuli (6, 17-19). cedures were exactly as described (30, 31). Neutralized HPLC Methylation is mediated by a protein methyltransferase en- fractions containing radiolabeled peptides K1 and Ri were pooled coded by the cheR gene and results in the formation of the y- and lyophilized directly without removal of sodium phosphate. The publication costs of this article were defrayed in part by page charge Abbreviation: MCP, methyl-accepting chemotaxis protein. payment. This article must therefore be hereby marked "advertise- t Present address: DNAX Research Institute of Molecular and Cellular ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Biology, Inc., 1450 Page Mill Rd., Palo Alto, CA 94304. 3599 Downloaded by guest on September 26, 2021 3600 Biochemistry: Kehry et aL Proc. Natl. Acad. Sci. USA 80 (1983) Radiolabeled Sequence Analysis. The sequences shown in RESULTS Fig. 1 were determined on a spinning cup sequenator with a Positions of Methyl-Accepting Sites in Tryptic Peptides from single cleavage program as described (35, 36). To minimize hy- MCPI. Methyl-accepting sites on the two methyl-accepting drolrsis of the methyl esters during sequence determinations tryptic peptides from MCPI, K1 and R1, have been extensively on [ H]methyl-labeled peptides, the sequenator was modified characterized with respect to their identity and number (30). to bypass the conversion flask; the anilinothiazolinone deriv- The methylated forms of peptides K1 and R1 have been des- atives were extracted from the cup with 1-chlorobutane/0. 1% ignated T1-T6 in order of elution from a reverse-phase HPLC dithiothreitol and collected. For each sequenator cycle, the column. Methylated peptide T4 corresponds to the dimethylat- fraction that contained the derivatized amino acid was dried ed form of peptide R1. Peptides T2, T3, T5, and T6 represent under argon and dissolved in 0.2 ml of acetonitrile. Radioac- the mono-, di-, tri-, and tetramethylated forms of CheB-mod- tivity in the entire fraction was determined by liquid scintil- ified peptide KI, respectively, and are not observed in MCPI lation counting. All samples were loaded in 0.15% NaDodSO4 produced in a cheB mutant background (30, 31). Peptide Ti is (Bio-Rad, twice recrystallized), extracted with 4 ml of 1-chlo- the monomethylated form of peptide K1 that does not contain robutane/0. 1% dithiothreitol, and double coupled with an ex- a CheB modification; peptide Ti is only observed as a major tended first cleavage time. Sequence analysis of [3S]methio- methylated form in MCPI produced in cheB cells and does not nine-labeled peptide K1 was performed in duplicate and yielded accept more than one methyl group (31). similar results both times. To localize the positions of the methyl-accepting sites in the The sequences shown in Fig. 2 were determined on a gas- amino acid sequences of the peptides, radiolabeled peptides phase sequenator as described (37). Samples were loaded with were isolated from MCPI synthesized in wild-type cells and 0.3 mg of sperm whale myoglobin in 1% NaDodSO4. To min- subjected to protein sequence determinations. Sequence anal- imize hydrolysis of the methyl esters, delivery of the 1 M HCl/ ysis was performed on [3H]methyl-labeled peptide K1 that was methanol was turned off, and after delivery of 1-chlorobutane CheB-modified (peptides T3 and T5 combined) and established the extracted anilinothiazolinone derivatives (=0.3 ml) were methylated residues at positions 3, 10, and perhaps 17 (Figs. collected. No wash was performed, and recovery was estimated 1 and 2). A methylated residue also was found at position 11 in to be 80%. For each sequenator cycle, the sample was trans- the second methyl-accepting peptide, R1, from MCPI. Deter- ferred to a scintillation vial and 4 ml of Liquifluor (New En- gland Nuclear) was added for determination of radioactivity by scintillation counting (50 min per vial). Sequenator lag, which is observed as a significant yield (>10%) of the amino acid from one cycle in the following cycle, resulted from the large quan- tity of sodium phosphate (pH 2.2) in the samples, which changed C6) the pH of the coupling buffer. Several [3H]methyl-labeled peptides were incubated for var- ious times under the conditions used for coupling in the se- quenator (pH 9.0 at 54°C). These base-catalyzed hydrolyses in- dicated that the half-life of the methyl esters averaged 7 hr. The C,, coupling time for each sequenator cycle was a total of 30 min in the spinning cup sequenator and 15 min in the gas-phase se- quenator.
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