Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Oct 15;287(Pt 2):533–543. doi: 10.1042/bj2870533

Specificity and affinity of binding of phosphate-containing compounds to CheY protein.

L Kar 1, P Z De Croos 1, S J Roman 1, P Matsumura 1, M E Johnson 1
PMCID: PMC1133197  PMID: 1332676

Abstract

1H- and 31P-n.m.r. have been used to study the interaction of the bacterial chemotaxis protein, CheY, with ATP and a variety of other phosphates in the presence and absence of bivalent metal ions. In the metal-bound conformation, CheY will bind nucleotide phosphates and phosphates in general, while in the metal-free conformation CheY loses its affinity for phosphates. In the presence of low concentrations of nitroxide-spin-labelled ATP (SL-ATP), specific proton resonances of metal-bound CheY are suppressed, indicating that ATP binds to a specific site on this metal-bound form of the protein. These studies also show that the same resonances are affected by the binding of SL-ATP and Mn2+, indicating that the phosphate- and metal-binding sites are close to each other and to Asp-57 (the site of phosphorylation in CheY). 1H- and 31P-n.m.r. studies using ATP, GTP, TTP, UTP, ADP, AMP and inorganic phosphates show that the binding is not specific for adenine, and does not involve the base directly, but is mediated primarily by the phosphate groups. Experiments with a phosphorylation mutant (Asp-13-->Asn) suggest that the observed phosphate binding and activation of CheY by phosphorylation may be related. Our results indicate that the conformational change and charge interactions brought about by the binding of a metal ion at the active site are required for CheY to interact with a phosphate. These studies also demonstrate the utility of spin-label-induced relaxation in conjunction with two-dimensional-n.m.r. measurements for exploring ligand-binding sites.

Full text

PDF
533

Selected References

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

  1. 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]
  2. Guillory R. J., Jeng S. J. Arylazido nucleotide analogs in a photoaffinity approach to receptor site labeling. Methods Enzymol. 1977;46:259–288. doi: 10.1016/s0076-6879(77)46029-4. [DOI] [PubMed] [Google Scholar]
  3. Gupta R. K., Benovic J. L., Rose Z. B. Magnetic resonance studies of the binding of ATP and cations to human hemoglobin. J Biol Chem. 1978 Sep 10;253(17):6165–6171. [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Hess J. F., Oosawa K., Matsumura P., Simon M. I. Protein phosphorylation is involved in bacterial chemotaxis. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7609–7613. doi: 10.1073/pnas.84.21.7609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kar L., Matsumura P., Johnson M. E. Bivalent-metal binding to CheY protein. Effect on protein conformation. Biochem J. 1992 Oct 15;287(Pt 2):521–531. doi: 10.1042/bj2870521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kosen P. A., Scheek R. M., Naderi H., Basus V. J., Manogaran S., Schmidt P. G., Oppenheimer N. J., Kuntz I. D. Two-dimensional 1H NMR of three spin-labeled derivatives of bovine pancreatic trypsin inhibitor. Biochemistry. 1986 May 6;25(9):2356–2364. doi: 10.1021/bi00357a009. [DOI] [PubMed] [Google Scholar]
  9. Kosen P. A. Spin labeling of proteins. Methods Enzymol. 1989;177:86–121. doi: 10.1016/0076-6879(89)77007-5. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Lukat G. S., Stock A. M., Stock J. B. Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis. Biochemistry. 1990 Jun 12;29(23):5436–5442. doi: 10.1021/bi00475a004. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Oosawa K., Hess J. F., Simon M. I. Mutants defective in bacterial chemotaxis show modified protein phosphorylation. Cell. 1988 Apr 8;53(1):89–96. doi: 10.1016/0092-8674(88)90490-4. [DOI] [PubMed] [Google Scholar]
  14. Shioi J. I., Galloway R. J., Niwano M., Chinnock R. E., Taylor B. L. Requirement of ATP in bacterial chemotaxis. J Biol Chem. 1982 Jul 25;257(14):7969–7975. [PubMed] [Google Scholar]
  15. Smith J. M., Rowsell E. H., Shioi J., Taylor B. L. Identification of a site of ATP requirement for signal processing in bacterial chemotaxis. J Bacteriol. 1988 Jun;170(6):2698–2704. doi: 10.1128/jb.170.6.2698-2704.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Streckenbach B., Schwarz D., Repke K. R. Analysis of phosphoryl transfer mechanism and catalytic centre geometries of transport ATPase by means of spin-labelled ATP. Biochim Biophys Acta. 1980 Sep 2;601(1):34–46. doi: 10.1016/0005-2736(80)90511-8. [DOI] [PubMed] [Google Scholar]
  17. Volz K., Matsumura P. Crystal structure of Escherichia coli CheY refined at 1.7-A resolution. J Biol Chem. 1991 Aug 15;266(23):15511–15519. doi: 10.2210/pdb3chy/pdb. [DOI] [PubMed] [Google Scholar]
  18. 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]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES