Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1995 Sep;4(9):1801–1814. doi: 10.1002/pro.5560040915

1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR.

V A Feher 1, J W Zapf 1, J A Hoch 1, F W Dahlquist 1, J M Whiteley 1, J Cavanagh 1
PMCID: PMC2143210  PMID: 8528078

Abstract

Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many sidechain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Alatossava T., Jütte H., Kuhn A., Kellenberger E. Manipulation of intracellular magnesium content in polymyxin B nonapeptide-sensitized Escherichia coli by ionophore A23187. J Bacteriol. 1985 Apr;162(1):413–419. doi: 10.1128/jb.162.1.413-419.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bax A., Ikura M. An efficient 3D NMR technique for correlating the proton and 15N backbone amide resonances with the alpha-carbon of the preceding residue in uniformly 15N/13C enriched proteins. J Biomol NMR. 1991 May;1(1):99–104. doi: 10.1007/BF01874573. [DOI] [PubMed] [Google Scholar]
  3. Bellsolell L., Prieto J., Serrano L., Coll M. Magnesium binding to the bacterial chemotaxis protein CheY results in large conformational changes involving its functional surface. J Mol Biol. 1994 May 13;238(4):489–495. doi: 10.1006/jmbi.1994.1308. [DOI] [PubMed] [Google Scholar]
  4. Bourret R. B., Drake S. K., Chervitz S. A., Simon M. I., Falke J. J. Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by 19F NMR and protein engineering. J Biol Chem. 1993 Jun 25;268(18):13089–13096. [PMC free article] [PubMed] [Google Scholar]
  5. Bruix M., Pascual J., Santoro J., Prieto J., Serrano L., Rico M. 1H- and 15N-NMR assignment and solution structure of the chemotactic Escherichia coli Che Y protein. Eur J Biochem. 1993 Aug 1;215(3):573–585. doi: 10.1111/j.1432-1033.1993.tb18068.x. [DOI] [PubMed] [Google Scholar]
  6. Burbulys D., Trach K. A., Hoch J. A. Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell. 1991 Feb 8;64(3):545–552. doi: 10.1016/0092-8674(91)90238-t. [DOI] [PubMed] [Google Scholar]
  7. Dingermann T., Nerke K., Marschalek R. Influence of different 5'-flanking sequences of tRNA genes on their in vivo transcription efficiencies in Saccharomyces cerevisiae. Eur J Biochem. 1987 Dec 30;170(1-2):217–224. doi: 10.1111/j.1432-1033.1987.tb13689.x. [DOI] [PubMed] [Google Scholar]
  8. Driscoll P. C., Clore G. M., Marion D., Wingfield P. T., Gronenborn A. M. Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy. Biochemistry. 1990 Apr 10;29(14):3542–3556. doi: 10.1021/bi00466a018. [DOI] [PubMed] [Google Scholar]
  9. Forman-Kay J. E., Gronenborn A. M., Kay L. E., Wingfield P. T., Clore G. M. Studies on the solution conformation of human thioredoxin using heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Biochemistry. 1990 Feb 13;29(6):1566–1572. doi: 10.1021/bi00458a030. [DOI] [PubMed] [Google Scholar]
  10. Hartel A. J., Lankhorst P. P., Altona C. Thermodynamics of stacking and of self-association of the dinucleoside monophosphate m2(6)A-U from proton NMR chemical shifts: differential concentration temperature profile method. Eur J Biochem. 1982 Dec 15;129(2):343–357. doi: 10.1111/j.1432-1033.1982.tb07057.x. [DOI] [PubMed] [Google Scholar]
  11. Ikura M., Kay L. E., Bax A. A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry. 1990 May 15;29(19):4659–4667. doi: 10.1021/bi00471a022. [DOI] [PubMed] [Google Scholar]
  12. Lowry D. F., Roth A. F., Rupert P. B., Dahlquist F. W., Moy F. J., Domaille P. J., Matsumura P. Signal transduction in chemotaxis. A propagating conformation change upon phosphorylation of CheY. J Biol Chem. 1994 Oct 21;269(42):26358–26362. [PubMed] [Google Scholar]
  13. 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]
  14. Marion D., Driscoll P. C., Kay L. E., Wingfield P. T., Bax A., Gronenborn A. M., Clore G. M. Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta. Biochemistry. 1989 Jul 25;28(15):6150–6156. doi: 10.1021/bi00441a004. [DOI] [PubMed] [Google Scholar]
  15. Moy F. J., Lowry D. F., Matsumura P., Dahlquist F. W., Krywko J. E., Domaille P. J. Assignments, secondary structure, global fold, and dynamics of chemotaxis Y protein using three- and four-dimensional heteronuclear (13C,15N) NMR spectroscopy. Biochemistry. 1994 Sep 6;33(35):10731–10742. doi: 10.1021/bi00201a022. [DOI] [PubMed] [Google Scholar]
  16. Muchmore D. C., McIntosh L. P., Russell C. B., Anderson D. E., Dahlquist F. W. Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods Enzymol. 1989;177:44–73. doi: 10.1016/0076-6879(89)77005-1. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Rance M., Sørensen O. W., Bodenhausen G., Wagner G., Ernst R. R., Wüthrich K. Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. Biochem Biophys Res Commun. 1983 Dec 16;117(2):479–485. doi: 10.1016/0006-291x(83)91225-1. [DOI] [PubMed] [Google Scholar]
  19. Richardson J. S., Getzoff E. D., Richardson D. C. The beta bulge: a common small unit of nonrepetitive protein structure. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2574–2578. doi: 10.1073/pnas.75.6.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Richardson J. S., Richardson D. C. Amino acid preferences for specific locations at the ends of alpha helices. Science. 1988 Jun 17;240(4859):1648–1652. doi: 10.1126/science.3381086. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Sockett H., Yamaguchi S., Kihara M., Irikura V. M., Macnab R. M. Molecular analysis of the flagellar switch protein FliM of Salmonella typhimurium. J Bacteriol. 1992 Feb;174(3):793–806. doi: 10.1128/jb.174.3.793-806.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stock A. M., Martinez-Hackert E., Rasmussen B. F., West A. H., Stock J. B., Ringe D., Petsko G. A. Structure of the Mg(2+)-bound form of CheY and mechanism of phosphoryl transfer in bacterial chemotaxis. Biochemistry. 1993 Dec 14;32(49):13375–13380. doi: 10.1021/bi00212a001. [DOI] [PubMed] [Google Scholar]
  24. Stock A. M., Mottonen J. M., Stock J. B., Schutt C. E. Three-dimensional structure of CheY, the response regulator of bacterial chemotaxis. Nature. 1989 Feb 23;337(6209):745–749. doi: 10.1038/337745a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Swanson R. V., Simon M. I. Signal transduction. Bringing the eukaryotes up to speed. Curr Biol. 1994 Mar 1;4(3):234–237. doi: 10.1016/s0960-9822(00)00052-x. [DOI] [PubMed] [Google Scholar]
  27. Trach K. A., Chapman J. W., Piggot P. J., Hoch J. A. Deduced product of the stage 0 sporulation gene spo0F shares homology with the Spo0A, OmpR, and SfrA proteins. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7260–7264. doi: 10.1073/pnas.82.21.7260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Volz K. Structural conservation in the CheY superfamily. Biochemistry. 1993 Nov 9;32(44):11741–11753. doi: 10.1021/bi00095a001. [DOI] [PubMed] [Google Scholar]
  30. Wüthrich K., Billeter M., Braun W. Polypeptide secondary structure determination by nuclear magnetic resonance observation of short proton-proton distances. J Mol Biol. 1984 Dec 15;180(3):715–740. doi: 10.1016/0022-2836(84)90034-2. [DOI] [PubMed] [Google Scholar]
  31. Zuiderweg E. R., Fesik S. W. Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a. Biochemistry. 1989 Mar 21;28(6):2387–2391. doi: 10.1021/bi00432a008. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES