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.
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.
Feher
V A
VA
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene 97403, USA.
Zapf
J W
JW
Hoch
J A
JA
Dahlquist
F W
FW
Whiteley
J M
JM
Cavanagh
J
J
D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D006859:Hydrogen / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D015398:Signal Transduction
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D009587:Nitrogen Isotopes; C080951:Spo0F protein, Bacillus subtilis; C000607606:cheY protein, E coli; D006859:Hydrogen; D008274:Magnesium