A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction

Background: SpoOF and SpoOB specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli. SpoOF belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis. SpoOB is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on SpoOF, information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems. Results: Structural analysis of a co-crystal of the SpoOF response regulator interacting with the SpoOB phosphotransferase of the phosphorelay signal transduction system of B. subtilis was carried out using X-ray crystallographic techniques, The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer, Upon complex formation, the SpoOB conformation remains unchanged. SpoOF also retains the overall conformation; however, two loops around the active site show significant deviations. Conclusions: The SpoOF-SpoOB 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 SpoOF residues making up the hydrophobic patch are very similar in ail 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 beta 4-alpha 4 loop.