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. 1995 Apr;177(8):2218–2221. doi: 10.1128/jb.177.8.2218-2221.1995

In vivo sulfhydryl modification of the ligand-binding site of Tsr, the Escherichia coli serine chemoreceptor.

T Iwama 1, I Kawagishi 1, S Gomi 1, M Homma 1, Y Imae 1
PMCID: PMC176870  PMID: 7721714

Abstract

The Escherichia coli chemoreceptor Tsr mediates an attractant response to serine. We substituted Cys for Thr-156, one of the residues involved in serine sensing. The mutant receptor Tsr-T156C retained serine- and repellent-sensing abilities. However, it lost serine-sensing ability when it was treated in vivo with sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). Serine protected Tsr-T156C from these reagents. We showed that [3H]NEM bound to Tsr-T156C and that binding decreased in the presence of serine. By pretreating cells with serine and cold NEM, Tsr-T156C was selectively labeled with radioactive NEM. These results are consistent with the location of Thr-156 in the serine-binding site. Chemical modification of the Tsr ligand-binding site provides a basis for simple purification and should assist further in vivo and in vitro investigations of this chemoreceptor protein.

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Selected References

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  1. Bardwell J. C., Lee J. O., Jander G., Martin N., Belin D., Beckwith J. A pathway for disulfide bond formation in vivo. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1038–1042. doi: 10.1073/pnas.90.3.1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bardwell J. C., McGovern K., Beckwith J. Identification of a protein required for disulfide bond formation in vivo. Cell. 1991 Nov 1;67(3):581–589. doi: 10.1016/0092-8674(91)90532-4. [DOI] [PubMed] [Google Scholar]
  3. Bourret R. B., Borkovich K. A., Simon M. I. Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu Rev Biochem. 1991;60:401–441. doi: 10.1146/annurev.bi.60.070191.002153. [DOI] [PubMed] [Google Scholar]
  4. Boyd A., Kendall K., Simon M. I. Structure of the serine chemoreceptor in Escherichia coli. Nature. 1983 Feb 17;301(5901):623–626. doi: 10.1038/301623a0. [DOI] [PubMed] [Google Scholar]
  5. Burrows G. G., Newcomer M. E., Hazelbauer G. L. Purification of receptor protein Trg by exploiting a property common to chemotactic transducers of Escherichia coli. J Biol Chem. 1989 Oct 15;264(29):17309–17315. [PubMed] [Google Scholar]
  6. Dailey F. E., Berg H. C. Mutants in disulfide bond formation that disrupt flagellar assembly in Escherichia coli. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1043–1047. doi: 10.1073/pnas.90.3.1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gardina P., Conway C., Kossman M., Manson M. Aspartate and maltose-binding protein interact with adjacent sites in the Tar chemotactic signal transducer of Escherichia coli. J Bacteriol. 1992 Mar;174(5):1528–1536. doi: 10.1128/jb.174.5.1528-1536.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gomi S., Lee L., Iwama T., Imae Y. Inhibition of aspartate chemotaxis of Escherichia coli by site-directed sulfhydryl modification of the receptor. J Biochem. 1993 Feb;113(2):208–213. doi: 10.1093/oxfordjournals.jbchem.a124027. [DOI] [PubMed] [Google Scholar]
  9. Hedblom M. L., Adler J. Chemotactic response of Escherichia coli to chemically synthesized amino acids. J Bacteriol. 1983 Sep;155(3):1463–1466. doi: 10.1128/jb.155.3.1463-1466.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hedblom M. L., Adler J. Genetic and biochemical properties of Escherichia coli mutants with defects in serine chemotaxis. J Bacteriol. 1980 Dec;144(3):1048–1060. doi: 10.1128/jb.144.3.1048-1060.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Imae Y., Oosawa K., Mizuno T., Kihara M., Macnab R. M. Phenol: a complex chemoeffector in bacterial chemotaxis. J Bacteriol. 1987 Jan;169(1):371–379. doi: 10.1128/jb.169.1.371-379.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jeffery C. J., Koshland D. E., Jr Three-dimensional structural model of the serine receptor ligand-binding domain. Protein Sci. 1993 Apr;2(4):559–566. doi: 10.1002/pro.5560020407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kamitani S., Akiyama Y., Ito K. Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme. EMBO J. 1992 Jan;11(1):57–62. doi: 10.1002/j.1460-2075.1992.tb05027.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kossmann M., Wolff C., Manson M. D. Maltose chemoreceptor of Escherichia coli: interaction of maltose-binding protein and the tar signal transducer. J Bacteriol. 1988 Oct;170(10):4516–4521. doi: 10.1128/jb.170.10.4516-4521.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lee L., Imae Y. Role of threonine residue 154 in ligand recognition of the tar chemoreceptor in Escherichia coli. J Bacteriol. 1990 Jan;172(1):377–382. doi: 10.1128/jb.172.1.377-382.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee L., Mizuno T., Imae Y. Thermosensing properties of Escherichia coli tsr mutants defective in serine chemoreception. J Bacteriol. 1988 Oct;170(10):4769–4774. doi: 10.1128/jb.170.10.4769-4774.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lin L. N., Li J., Brandts J. F., Weis R. M. The serine receptor of bacterial chemotaxis exhibits half-site saturation for serine binding. Biochemistry. 1994 May 31;33(21):6564–6570. doi: 10.1021/bi00187a025. [DOI] [PubMed] [Google Scholar]
  18. Maeda K., Imae Y., Shioi J. I., Oosawa F. Effect of temperature on motility and chemotaxis of Escherichia coli. J Bacteriol. 1976 Sep;127(3):1039–1046. doi: 10.1128/jb.127.3.1039-1046.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Manoil C., Beckwith J. A genetic approach to analyzing membrane protein topology. Science. 1986 Sep 26;233(4771):1403–1408. doi: 10.1126/science.3529391. [DOI] [PubMed] [Google Scholar]
  20. Manson M. D. Bacterial motility and chemotaxis. Adv Microb Physiol. 1992;33:277–346. doi: 10.1016/s0065-2911(08)60219-2. [DOI] [PubMed] [Google Scholar]
  21. Milburn M. V., Privé G. G., Milligan D. L., Scott W. G., Yeh J., Jancarik J., Koshland D. E., Jr, Kim S. H. Three-dimensional structures of the ligand-binding domain of the bacterial aspartate receptor with and without a ligand. Science. 1991 Nov 29;254(5036):1342–1347. doi: 10.1126/science.1660187. [DOI] [PubMed] [Google Scholar]
  22. Missiakas D., Georgopoulos C., Raina S. Identification and characterization of the Escherichia coli gene dsbB, whose product is involved in the formation of disulfide bonds in vivo. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7084–7088. doi: 10.1073/pnas.90.15.7084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mowbray S. L., Koshland D. E., Jr Mutations in the aspartate receptor of Escherichia coli which affect aspartate binding. J Biol Chem. 1990 Sep 15;265(26):15638–15643. [PubMed] [Google Scholar]
  24. Oosawa K., Imae Y. Glycerol and ethylene glycol: members of a new class of repellents of Escherichia coli chemotaxis. J Bacteriol. 1983 Apr;154(1):104–112. doi: 10.1128/jb.154.1.104-112.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Parkinson J. S. Signal transduction schemes of bacteria. Cell. 1993 Jun 4;73(5):857–871. doi: 10.1016/0092-8674(93)90267-t. [DOI] [PubMed] [Google Scholar]
  26. Rice M. S., Dahlquist F. W. Sites of deamidation and methylation in Tsr, a bacterial chemotaxis sensory transducer. J Biol Chem. 1991 May 25;266(15):9746–9753. [PubMed] [Google Scholar]
  27. Scott W. G., Milligan D. L., Milburn M. V., Privé G. G., Yeh J., Koshland D. E., Jr, Kim S. H. Refined structures of the ligand-binding domain of the aspartate receptor from Salmonella typhimurium. J Mol Biol. 1993 Jul 20;232(2):555–573. doi: 10.1006/jmbi.1993.1411. [DOI] [PubMed] [Google Scholar]
  28. Springer M. S., Goy M. F., Adler J. Sensory transduction in Escherichia coli: two complementary pathways of information processing that involve methylated proteins. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3312–3316. doi: 10.1073/pnas.74.8.3312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stock J. B., Surette M. G., McCleary W. R., Stock A. M. Signal transduction in bacterial chemotaxis. J Biol Chem. 1992 Oct 5;267(28):19753–19756. [PubMed] [Google Scholar]
  30. Tatsuno I., Lee L., Kawagishi I., Homma M., Imae Y. Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane-spanning regions. Mol Microbiol. 1994 Nov;14(4):755–762. doi: 10.1111/j.1365-2958.1994.tb01312.x. [DOI] [PubMed] [Google Scholar]
  31. Tsang N., Macnab R., Koshland D. E., Jr Common mechanism for repellents and attractants in bacterial chemotaxis. Science. 1973 Jul 6;181(4094):60–63. doi: 10.1126/science.181.4094.60. [DOI] [PubMed] [Google Scholar]
  32. Tso W. W., Adler J. Negative chemotaxis in Escherichia coli. J Bacteriol. 1974 May;118(2):560–576. doi: 10.1128/jb.118.2.560-576.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wolfe A. J., Conley M. P., Kramer T. J., Berg H. C. Reconstitution of signaling in bacterial chemotaxis. J Bacteriol. 1987 May;169(5):1878–1885. doi: 10.1128/jb.169.5.1878-1885.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wolff C., Parkinson J. S. Aspartate taxis mutants of the Escherichia coli tar chemoreceptor. J Bacteriol. 1988 Oct;170(10):4509–4515. doi: 10.1128/jb.170.10.4509-4515.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yeh J. I., Biemann H. P., Pandit J., Koshland D. E., Kim S. H. The three-dimensional structure of the ligand-binding domain of a wild-type bacterial chemotaxis receptor. Structural comparison to the cross-linked mutant forms and conformational changes upon ligand binding. J Biol Chem. 1993 May 5;268(13):9787–9792. [PubMed] [Google Scholar]

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