The structure of a binary complex of dihy-

The four individual phosphotransfer steps in the multicomponent phosphorelay system controlling sporulation in Bacillus subtilis have been characterized kinetically using highly purified samples of the individual protein components in vitro. The autophosphorylation of KinA is the initial occurrence, and a divalent metal ion is required. KinA-mediated phosphotransfer, which displays a 57,000-fold preference (kcat/Km) for catalysis of Spo0F-P formation relative to Spo0A-P formation, is shown to proceed via a hybrid ping-pong/sequential mechanism with pronounced (> or = 40-fold) substrate synergism by Spo0F of KinA autophosphorylation. In addition, evidence is presented for formation of an abortive KinA.Spo0F complex. Kinetic parameters derived for Spo0F-P and Spo0A as substrates for Spo0B, the second phosphotransferase in the phosphorelay chain, indicate that Spo0B-mediated production of Spo0A-P is 1.1-million-fold more efficient (kcat/KSpo0A) than the direct KinA-mediated process. A rationale is presented for a four component cascade as the means for controlling sporulation, which focuses on the utility of synergistic interactions among the phosphorelay components that may be modulated by environmental stimuli.

The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

A basis for understanding specificity of molecular recognition between phosphorelay proteins has been deduced from the 2.6 A structure of the Spo0B phosphotransferase of the phosphorelay regulating sporulation initiation. Spo0B consists of two domains: an N-terminal alpha-helical hairpin domain and a C-terminal alpha/beta domain. Two subunits of Spo0B dimerize by a parallel association of helical hairpins to form a novel four-helix bundle from which the active histidine protrudes. Docking studies show that both the monomers interact with a Spo0F molecule at the region surrounding the active site aspartate to position it for phosphotransfer. It is apparent that different surfaces of response regulators may be involved in recognition of the protein partners to which they are paired.

NMR has been employed for structural and dynamic studies of the bacterial response regulator, Spo0F. This 124-residue protein is an essential component of the sporulation phosphorelay signal transduction pathway in Bacillus subtilis. Three-dimensional 1H, 15N, and 13C experiments have been used to obtain full side chain assignments and the 1511 distance, 121 dihedral angle, and 80 hydrogen bonding restraints required for generating a family of structures (14 restraints per residue). The structures give a well-defined (alpha/beta)5 fold for residues 4-120 with average rms deviations of 0.59 A for backbone heavy atoms and 1.02 A for all heavy atoms. Analyses of backbone 15N relaxation measurements demonstrate relative rigidity in most regions of regular secondary structure with a generalized order parameter (S2) of 0.9 +/- 0.05 and a rotational correlation time (taum) of 7.0 +/- 0.5 ns. Loop regions near the site of phosphorylation have higher than average rms deviation values and T1/T2 ratios suggesting significant internal motion or chemical exchange at these sites. Additionally, multiple conformers are observed for the beta4-alpha4 loop and beta-strand 5 region. These conformers may be related to structural changes associated with phosphorylation and also indicative of the propensity this recognition surface has for differential protein interactions. Comparison of Spo0F structural features to those of other response regulators reveals subtle differences in the orientations of secondary structure in the putative recognition surfaces and the relative charge distribution of residues surrounding the site of phosphorylation. These may be important in providing specificity for protein-protein interactions and for determining the lifetimes of the phosphorylated state.

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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.

Transient phosphorylation at an aspartate residue on the Spo0F protein is a central step in the phosphorelay signal transduction pathway controlling sporulation in Bacilli. The response regulator Spo0F-P is stable to hydrolysis (t1/2 > 24 h at 23 degrees C in the absence of Mg2+), allowing the use of nondenaturing PAGE to separate the phosphorylated and non-phosphorylated forms of Spo0F. Using this novel assay, phosphoramidate containing compounds were found to specifically phosphorylate Spo0F, a reaction that requires the presence of a divalent metal, but mixed phosphate-carboxylate compounds did not act as phospho donors. Rapid hydrolysis of Spo0F-P generated with phosphoramidate by proteins downstream in the phosphorelay (Spo0B and Spo0A) is consistent with phosphorylation at the active site of Spo0F. The initial rate of Spo0F-P formation from phosphoramidate displays Michaelis-Menten kinetics, providing evidence for the proposal that response regulators, such as Spo0F, function as phosphoryl transferase enzymes (McCleary et al., 1993). The results establish that Spo0F functions as a phosphoryl transferase that uses exclusively a phosphoramidate rather than an acyl phosphate as substrate during autophosphorylation.