1H,15N, and13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of thebacillus subtilisresponse regulator, SpoOF, determined by heteronuclear high-resolution NMR

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Abstract
SpoOF, 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 side‐chain proton resonances are also reported. The secondary structure, inferred from short‐ and medium‐range NOEs, 3JHNα coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (α/β)5 fold. Interestingly, comparison of the secondary structure of SpoOF 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 2/5:573–585), 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 SpoOF, 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.