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

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.

Crystallographic and Biochemical Studies of DivK Reveal Novel Features of an Essential Response Regulator in Caulobacter crescentus

“…One of the water molecules was hydrogen bonded to the main chain carbonyl oxygen of Gln-55. The coordination of the metal to the third acidic residue (Glu-9) and the absence of direct coordination to the main chain carbonyl oxygen of Gln-55 differs from what has been reported for all metal-bound receiver domains (32)(33)(34)(35)(36).…”

A Role for Asp75 in Domain Interactions in theBacillus subtilis Response Regulator Spo0A

“…These response regulators are activated by phosphorylation of their regulatory domain (11,12). The regulatory domains have a structure similar to that of the single domain response regulators CheY (13,14) and Spo0F (15,16) including the key conserved amino acids known to be important for signal propagation in CheY (reviewed in Refs. 14 and 17).…”

The Crystal Structure of the Phosphorylation Domain in PhoP Reveals a Functional Tandem Association Mediated by an Asymmetric Interface

“…The structural similarities among the effector modules in OmpR, PhoB, and DrrD (24,29,38,45) and among regulatory domains in general (2,7,13,18,19,23,32,37,41,48,49,51,52) are in sharp contrast to the different strategies that are used in the response regulators to regulate protein activity and to respond to the common phosphorylation event. The spatial extent and magnitude of the conformational changes in the ␣4-␤5-␣5 region that essentially sense phosphorylation of the aspartic acid vary in the few regulatory domains that have been characterized in both states (3,10,20,28,31).…”