H-1, N-15, AND C-13 BACKBONE CHEMICAL-SHIFT ASSIGNMENTS, SECONDARY STRUCTURE, AND MAGNESIUM-BINDING CHARACTERISTICS OF THE BACILLUS-SUBTILIS RESPONSE REGULATOR, SPOOF, 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 H-1, N-15, and C-13 chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, N-15 TOCSY-HSQC, N-15 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, (3)J(HN 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) (Volt K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, fur 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.