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. 1995 Oct;6(10):1367–1380. doi: 10.1091/mbc.6.10.1367

Computer analysis of the binding reactions leading to a transmembrane receptor-linked multiprotein complex involved in bacterial chemotaxis.

D Bray 1, R B Bourret 1
PMCID: PMC301293  PMID: 8573792

Abstract

The chemotactic response of bacteria is mediated by complexes containing two molecules each of a transmembrane receptor and the intracellular signaling proteins CheA and CheW. Mutants in which one or the other of the proteins of this complex are absent, inactive, or expressed at elevated amounts show altered chemotactic behavior and the phenotypes are difficult to interpret for some overexpression mutants. We have examined the possibility that these unexpected phenotypes might arise from the binding steps that lead to active complex formation. A limited genetic algorithm was used to search for sets of binding reactions and associated binding constants expected to give mutant phenotypes in accord with experimental data. Different sets of binding equilibria and different assumptions about the activity of particular receptor complexes were tried. Computer analysis demonstrated that it is possible to obtain sets of binding equilibria consistent with the observed phenotypes and provided a simple explanation for these phenotypes in terms of the distribution of active and inactive complexes formed under various conditions. Optimization methods of this kind offer a unique way to analyze reactions taking place inside living cells based on behavioral data.

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Selected References

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

  1. Ames P., Parkinson J. S. Constitutively signaling fragments of Tsr, the Escherichia coli serine chemoreceptor. J Bacteriol. 1994 Oct;176(20):6340–6348. doi: 10.1128/jb.176.20.6340-6348.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Block S. M., Segall J. E., Berg H. C. Impulse responses in bacterial chemotaxis. Cell. 1982 Nov;31(1):215–226. doi: 10.1016/0092-8674(82)90421-4. [DOI] [PubMed] [Google Scholar]
  4. Bochner B. R., Ames B. N. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J Biol Chem. 1982 Aug 25;257(16):9759–9769. [PubMed] [Google Scholar]
  5. Borkovich K. A., Alex L. A., Simon M. I. Attenuation of sensory receptor signaling by covalent modification. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6756–6760. doi: 10.1073/pnas.89.15.6756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. Bray D., Bourret R. B., Simon M. I. Computer simulation of the phosphorylation cascade controlling bacterial chemotaxis. Mol Biol Cell. 1993 May;4(5):469–482. doi: 10.1091/mbc.4.5.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bray D., Lay S. Computer simulated evolution of a network of cell-signaling molecules. Biophys J. 1994 Apr;66(4):972–977. doi: 10.1016/S0006-3495(94)80878-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bray D., Lay S. Rapid numerical integration algorithm for finding the equilibrium state of a system of coupled binding reactions. Comput Appl Biosci. 1994 Sep;10(5):471–476. doi: 10.1093/bioinformatics/10.5.471. [DOI] [PubMed] [Google Scholar]
  13. Choi K. Y., Satterberg B., Lyons D. M., Elion E. A. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell. 1994 Aug 12;78(3):499–512. doi: 10.1016/0092-8674(94)90427-8. [DOI] [PubMed] [Google Scholar]
  14. Clapham D. E., Neer E. J. New roles for G-protein beta gamma-dimers in transmembrane signalling. Nature. 1993 Sep 30;365(6445):403–406. doi: 10.1038/365403a0. [DOI] [PubMed] [Google Scholar]
  15. Cohen G. B., Ren R., Baltimore D. Modular binding domains in signal transduction proteins. Cell. 1995 Jan 27;80(2):237–248. doi: 10.1016/0092-8674(95)90406-9. [DOI] [PubMed] [Google Scholar]
  16. Conklin B. R., Bourne H. R. Structural elements of G alpha subunits that interact with G beta gamma, receptors, and effectors. Cell. 1993 May 21;73(4):631–641. doi: 10.1016/0092-8674(93)90245-l. [DOI] [PubMed] [Google Scholar]
  17. DeFranco A. L., Koshland D. E., Jr Molecular cloning of chemotaxis genes and overproduction of gene products in the bacterial sensing system. J Bacteriol. 1981 Aug;147(2):390–400. doi: 10.1128/jb.147.2.390-400.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gegner J. A., Dahlquist F. W. Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):750–754. doi: 10.1073/pnas.88.3.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hess J. F., Bourret R. B., Oosawa K., Matsumura P., Simon M. I. Protein phosphorylation and bacterial chemotaxis. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 1):41–48. doi: 10.1101/sqb.1988.053.01.008. [DOI] [PubMed] [Google Scholar]
  21. Hess J. F., Oosawa K., Kaplan N., Simon M. I. Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis. Cell. 1988 Apr 8;53(1):79–87. doi: 10.1016/0092-8674(88)90489-8. [DOI] [PubMed] [Google Scholar]
  22. Kuo S. C., Koshland D. E., Jr Multiple kinetic states for the flagellar motor switch. J Bacteriol. 1989 Nov;171(11):6279–6287. doi: 10.1128/jb.171.11.6279-6287.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Lukat G. S., Lee B. H., Mottonen J. M., Stock A. M., Stock J. B. Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis. J Biol Chem. 1991 May 5;266(13):8348–8354. [PubMed] [Google Scholar]
  26. Lukat G. S., McCleary W. R., Stock A. M., Stock J. B. Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):718–722. doi: 10.1073/pnas.89.2.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Maddock J. R., Shapiro L. Polar location of the chemoreceptor complex in the Escherichia coli cell. Science. 1993 Mar 19;259(5102):1717–1723. doi: 10.1126/science.8456299. [DOI] [PubMed] [Google Scholar]
  29. Marcus S., Polverino A., Barr M., Wigler M. Complexes between STE5 and components of the pheromone-responsive mitogen-activated protein kinase module. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7762–7766. doi: 10.1073/pnas.91.16.7762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McNally D. F., Matsumura P. Bacterial chemotaxis signaling complexes: formation of a CheA/CheW complex enhances autophosphorylation and affinity for CheY. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6269–6273. doi: 10.1073/pnas.88.14.6269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Milligan D. L., Koshland D. E., Jr Site-directed cross-linking. Establishing the dimeric structure of the aspartate receptor of bacterial chemotaxis. J Biol Chem. 1988 May 5;263(13):6268–6275. [PubMed] [Google Scholar]
  32. Mochly-Rosen D. Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science. 1995 Apr 14;268(5208):247–251. doi: 10.1126/science.7716516. [DOI] [PubMed] [Google Scholar]
  33. Nakayama T., Wiest D. L., Abraham K. M., Munitz T. I., Perlmutter R. M., Singer A. Decreased signaling competence as a result of receptor overexpression: overexpression of CD4 reduces its ability to activate p56lck tyrosine kinase and to regulate T-cell antigen receptor expression in immature CD4+CD8+ thymocytes. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10534–10538. doi: 10.1073/pnas.90.22.10534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ninfa E. G., Stock A., Mowbray S., Stock J. Reconstitution of the bacterial chemotaxis signal transduction system from purified components. J Biol Chem. 1991 May 25;266(15):9764–9770. [PubMed] [Google Scholar]
  35. Northrup S. H., Erickson H. P. Kinetics of protein-protein association explained by Brownian dynamics computer simulation. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3338–3342. doi: 10.1073/pnas.89.8.3338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Parkinson J. S. Complementation analysis and deletion mapping of Escherichia coli mutants defective in chemotaxis. J Bacteriol. 1978 Jul;135(1):45–53. doi: 10.1128/jb.135.1.45-53.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pawson T. Protein modules and signalling networks. Nature. 1995 Feb 16;373(6515):573–580. doi: 10.1038/373573a0. [DOI] [PubMed] [Google Scholar]
  38. Pawson T., Schlessingert J. SH2 and SH3 domains. Curr Biol. 1993 Jul 1;3(7):434–442. doi: 10.1016/0960-9822(93)90350-w. [DOI] [PubMed] [Google Scholar]
  39. Prüss B. M., Wolfe A. J. Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli. Mol Microbiol. 1994 Jun;12(6):973–984. doi: 10.1111/j.1365-2958.1994.tb01085.x. [DOI] [PubMed] [Google Scholar]
  40. Sanders D. A., Mendez B., Koshland D. E., Jr Role of the CheW protein in bacterial chemotaxis: overexpression is equivalent to absence. J Bacteriol. 1989 Nov;171(11):6271–6278. doi: 10.1128/jb.171.11.6271-6278.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schuster S. C., Swanson R. V., Alex L. A., Bourret R. B., Simon M. I. Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance. Nature. 1993 Sep 23;365(6444):343–347. doi: 10.1038/365343a0. [DOI] [PubMed] [Google Scholar]
  42. Stewart R. C. Activating and inhibitory mutations in the regulatory domain of CheB, the methylesterase in bacterial chemotaxis. J Biol Chem. 1993 Jan 25;268(3):1921–1930. [PubMed] [Google Scholar]
  43. Stock A., Koshland D. E., Jr, Stock J. Homologies between the Salmonella typhimurium CheY protein and proteins involved in the regulation of chemotaxis, membrane protein synthesis, and sporulation. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7989–7993. doi: 10.1073/pnas.82.23.7989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stock J. B., Lukat G. S., Stock A. M. Bacterial chemotaxis and the molecular logic of intracellular signal transduction networks. Annu Rev Biophys Biophys Chem. 1991;20:109–136. doi: 10.1146/annurev.bb.20.060191.000545. [DOI] [PubMed] [Google Scholar]
  45. Swanson R. V., Bourret R. B., Simon M. I. Intermolecular complementation of the kinase activity of CheA. Mol Microbiol. 1993 May;8(3):435–441. doi: 10.1111/j.1365-2958.1993.tb01588.x. [DOI] [PubMed] [Google Scholar]
  46. Tawa P., Stewart R. C. Kinetics of CheA autophosphorylation and dephosphorylation reactions. Biochemistry. 1994 Jun 28;33(25):7917–7924. doi: 10.1021/bi00191a019. [DOI] [PubMed] [Google Scholar]
  47. Welch M., Oosawa K., Aizawa S. I., Eisenbach M. Effects of phosphorylation, Mg2+, and conformation of the chemotaxis protein CheY on its binding to the flagellar switch protein FliM. Biochemistry. 1994 Aug 30;33(34):10470–10476. doi: 10.1021/bi00200a031. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Welch P. J., Wang J. Y. Disruption of retinoblastoma protein function by coexpression of its C pocket fragment. Genes Dev. 1995 Jan 1;9(1):31–46. doi: 10.1101/gad.9.1.31. [DOI] [PubMed] [Google Scholar]
  50. Wolfe A. J., McNamara B. P., Stewart R. C. The short form of CheA couples chemoreception to CheA phosphorylation. J Bacteriol. 1994 Aug;176(15):4483–4491. doi: 10.1128/jb.176.15.4483-4491.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wylie D., Stock A., Wong C. Y., Stock J. Sensory transduction in bacterial chemotaxis involves phosphotransfer between Che proteins. Biochem Biophys Res Commun. 1988 Mar 15;151(2):891–896. doi: 10.1016/s0006-291x(88)80365-6. [DOI] [PubMed] [Google Scholar]
  52. Zimmerman S. B., Minton A. P. Macromolecular crowding: biochemical, biophysical, and physiological consequences. Annu Rev Biophys Biomol Struct. 1993;22:27–65. doi: 10.1146/annurev.bb.22.060193.000331. [DOI] [PubMed] [Google Scholar]
  53. Zimmerman S. B., Trach S. O. Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J Mol Biol. 1991 Dec 5;222(3):599–620. doi: 10.1016/0022-2836(91)90499-v. [DOI] [PubMed] [Google Scholar]

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