The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions. The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins. The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs. The bulk of the interactions outside this patch are through nonconserved residues. Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.

Sporulation in Bacillus species, the ultimate bacterial adaptive response, requires the precisely coordinated expression of a complex genetic pathway, and is initiated through the accumulation of the phosphorylated form of Spo0A, a pleiotropic response regulator transcription factor. Spo0A controls the transcription of several hundred genes in all spore-forming Bacilli including genes for sporulation and toxin regulation in pathogens such as Bacillus anthracis. The crystal structure of the effector domain of Spo0A from Bacillus subtilis in complex with its DNA target was determined. In the crystal lattice, two molecules form a tandem dimer upon binding to adjacent sites on DNA. The protein:protein and protein:DNA interfaces revealed in the crystal provide a basis for interpreting the transcription activation process and for the design of drugs to counter infections by these bacteria.

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.

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.