Phosphotransfer reactions of the CbbRRS three-protein two- component system from Rhodopseudomonas palustris CGA010 appear to be controlled by an internal molecular switch on the sensor kinase.

The CbbRRS system is an atypical three-protein two-component system that modulates the expression of the cbb(I) CO(2) fixation operon of Rhodopseudomonas palustris, possibly in response to a redox signal. It consists of a membrane-bound hybrid sensor kinase, CbbSR, with a transmitter and receiver domain, and two response regulator proteins, CbbRR1 and CbbRR2. No detectable helix-turn-helix DNA binding domain is associated with either response regulator, but an HPt domain and a second receiver domain are predicted at the C-terminal region of CbbRR1 and CbbRR2, respectively. The abundance of conserved residues predicted to participate in a His-Asp phosphorelay raised the question of their de facto involvement. In this study, the role of the multiple receiver domains was elucidated in vitro by generating site-directed mutants of the putative conserved residues. Distinct phosphorylation patterns were obtained with two truncated versions of the hybrid sensor kinase, CbbSR(T189) and CbbSR(R96) (CbbSR beginning at residues T189 and R96, respectively). These constructs also exhibited substantially different affinities for ATP and phosphorylation stability, which was found to be dependent on a conserved Asp residue (Asp-696) within the kinase receiver domain. Asp-696 also played an important role in defining the specificity of phosphorylation for response regulators CbbRR1 or CbbRR2, and this residue appeared to act in conjunction with residues within the region from Arg-96 to Thr-189 at the N terminus of the sensor kinase. The net effect of concerted interactions at these distinct regions of CbbSR created an internal molecular switch that appears to coordinate a unique branched phosphorelay system.