Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Jun 7;91(12):5485–5489. doi: 10.1073/pnas.91.12.5485

Liberation of an interaction domain from the phosphotransfer region of CheA, a signaling kinase of Escherichia coli.

T B Morrison 1, J S Parkinson 1
PMCID: PMC44020  PMID: 8202513

Abstract

The CheA protein of Escherichia coli is a histidine autokinase that donates its phosphate groups to two target proteins, CheY and CheB, to regulate flagellar rotation and sensory adaptation during chemotactic responses. The amino-terminal third of CheA contains the autophosphorylation site, determinants needed to interact with the catalytic center of the molecule, and determinants needed for specific recognition of its phosphorylation targets. To understand the structural basis for these activities, we examined the domain organization of the CheA phosphotransfer region by using DNA sequence analysis, limited proteolytic digestion, and a genetic technique called domain liberation. Comparison of the functionally interchangeable CheA proteins of E. coli and Salmonella typhimurium revealed two extensively mismatched segments within the phosphotransfer region, 22 and 25 aa long, with sequences characteristic of domain linkers. Both segments were readily susceptible to proteases, implying that they have an extended, flexible structure. In contrast, the intervening segments of the phosphotransfer region, designated P1 and P2 (roughly 140 and 65 aa, respectively), were relatively insensitive, suggesting they correspond to more compactly folded structural domains. Their functional properties were explored by identifying portions of the cheA coding region capable of interfering with chemotactic behavior when "liberated" and expressed as polypeptides. P1 fragments were not inhibitory, but P2 fragments blocked the interaction of CheY with the rotational switch at the flagellar motor, leading to incessant forward swimming. These results suggest that P2 contains CheY-binding determinants which are normally responsible for phosphotransfer specificity. Domain-liberation approaches should prove generally useful for analyzing multidomain proteins and their interaction targets.

Full text

PDF
5485

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. Ames P., Parkinson J. S. Transmembrane signaling by bacterial chemoreceptors: E. coli transducers with locked signal output. Cell. 1988 Dec 2;55(5):817–826. doi: 10.1016/0092-8674(88)90137-7. [DOI] [PubMed] [Google Scholar]
  3. Barak R., Eisenbach M. Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor. Biochemistry. 1992 Feb 18;31(6):1821–1826. doi: 10.1021/bi00121a034. [DOI] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Borkovich K. A., Kaplan N., Hess J. F., Simon M. I. Transmembrane signal transduction in bacterial chemotaxis involves ligand-dependent activation of phosphate group transfer. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1208–1212. doi: 10.1073/pnas.86.4.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Borkovich K. A., Simon M. I. The dynamics of protein phosphorylation in bacterial chemotaxis. Cell. 1990 Dec 21;63(6):1339–1348. doi: 10.1016/0092-8674(90)90429-i. [DOI] [PubMed] [Google Scholar]
  7. Bourret R. B., Davagnino J., Simon M. I. The carboxy-terminal portion of the CheA kinase mediates regulation of autophosphorylation by transducer and CheW. J Bacteriol. 1993 Apr;175(7):2097–2101. doi: 10.1128/jb.175.7.2097-2101.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bourret R. B., Hess J. F., Simon M. I. Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY. Proc Natl Acad Sci U S A. 1990 Jan;87(1):41–45. doi: 10.1073/pnas.87.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Callahan A. M., Frazier B. L., Parkinson J. S. Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage. J Bacteriol. 1987 Mar;169(3):1246–1253. doi: 10.1128/jb.169.3.1246-1253.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeFranco A. L., Parkinson J. S., Koshland D. E., Jr Functional homology of chemotaxis genes in Escherichia coli and Salmonella typhimurium. J Bacteriol. 1979 Jul;139(1):107–114. doi: 10.1128/jb.139.1.107-114.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gegner J. A., Graham D. R., Roth A. F., Dahlquist F. W. Assembly of an MCP receptor, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway. Cell. 1992 Sep 18;70(6):975–982. doi: 10.1016/0092-8674(92)90247-a. [DOI] [PubMed] [Google Scholar]
  13. Hess J. F., Bourret R. B., Simon M. I. Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis. Nature. 1988 Nov 10;336(6195):139–143. doi: 10.1038/336139a0. [DOI] [PubMed] [Google Scholar]
  14. Kofoid E. C., Parkinson J. S. Tandem translation starts in the cheA locus of Escherichia coli. J Bacteriol. 1991 Mar;173(6):2116–2119. doi: 10.1128/jb.173.6.2116-2119.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kuo S. C., Koshland D. E., Jr Roles of cheY and cheZ gene products in controlling flagellar rotation in bacterial chemotaxis of Escherichia coli. J Bacteriol. 1987 Mar;169(3):1307–1314. doi: 10.1128/jb.169.3.1307-1314.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liu J. D., Parkinson J. S. Genetics and sequence analysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control. J Bacteriol. 1989 Mar;171(3):1254–1261. doi: 10.1128/jb.171.3.1254-1261.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu J. D., Parkinson J. S. Role of CheW protein in coupling membrane receptors to the intracellular signaling system of bacterial chemotaxis. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8703–8707. doi: 10.1073/pnas.86.22.8703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lupas A., Stock J. Phosphorylation of an N-terminal regulatory domain activates the CheB methylesterase in bacterial chemotaxis. J Biol Chem. 1989 Oct 15;264(29):17337–17342. [PubMed] [Google Scholar]
  19. Matsumura P., Rydel J. J., Linzmeier R., Vacante D. Overexpression and sequence of the Escherichia coli cheY gene and biochemical activities of the CheY protein. J Bacteriol. 1984 Oct;160(1):36–41. doi: 10.1128/jb.160.1.36-41.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Park C., Hazelbauer G. L. Mutations specifically affecting ligand interaction of the Trg chemosensory transducer. J Bacteriol. 1986 Jul;167(1):101–109. doi: 10.1128/jb.167.1.101-109.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Parkinson J. S., Houts S. E. Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions. J Bacteriol. 1982 Jul;151(1):106–113. doi: 10.1128/jb.151.1.106-113.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Parkinson J. S., Kofoid E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet. 1992;26:71–112. doi: 10.1146/annurev.ge.26.120192.000443. [DOI] [PubMed] [Google Scholar]
  23. Parkinson J. S., Parker S. R., Talbert P. B., Houts S. E. Interactions between chemotaxis genes and flagellar genes in Escherichia coli. J Bacteriol. 1983 Jul;155(1):265–274. doi: 10.1128/jb.155.1.265-274.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Parkinson J. S. cheA, cheB, and cheC genes of Escherichia coli and their role in chemotaxis. J Bacteriol. 1976 May;126(2):758–770. doi: 10.1128/jb.126.2.758-770.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roman S. J., Meyers M., Volz K., Matsumura P. A chemotactic signaling surface on CheY defined by suppressors of flagellar switch mutations. J Bacteriol. 1992 Oct;174(19):6247–6255. doi: 10.1128/jb.174.19.6247-6255.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Slocum M. K., Parkinson J. S. Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: organization of the tar region. J Bacteriol. 1983 Aug;155(2):565–577. doi: 10.1128/jb.155.2.565-577.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Smith R. A., Parkinson J. S. Overlapping genes at the cheA locus of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5370–5374. doi: 10.1073/pnas.77.9.5370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stock A., Chen T., Welsh D., Stock J. CheA protein, a central regulator of bacterial chemotaxis, belongs to a family of proteins that control gene expression in response to changing environmental conditions. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1403–1407. doi: 10.1073/pnas.85.5.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Swanson R. V., Schuster S. C., Simon M. I. Expression of CheA fragments which define domains encoding kinase, phosphotransfer, and CheY binding activities. Biochemistry. 1993 Aug 3;32(30):7623–7629. doi: 10.1021/bi00081a004. [DOI] [PubMed] [Google Scholar]
  31. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  32. Welch M., Oosawa K., Aizawa S., Eisenbach M. Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8787–8791. doi: 10.1073/pnas.90.19.8787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wolfe A. J., Conley M. P., Berg H. C. Acetyladenylate plays a role in controlling the direction of flagellar rotation. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6711–6715. doi: 10.1073/pnas.85.18.6711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wootton J. C., Drummond M. H. The Q-linker: a class of interdomain sequences found in bacterial multidomain regulatory proteins. Protein Eng. 1989 May;2(7):535–543. doi: 10.1093/protein/2.7.535. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES