Genetic evidence that the alpha 5 helix of the receiver domain of PhoB is involved in interdomain interactions

Two-component signaling proteins are involved in transducing environmental stimuli into intracellular signals. Information is transmitted through a ph osphorylation cascade that consists of a histidine protein kinase and a res ponse regulator protein. Generally, response regulators are made up of a re ceiver domain and an output domain. Phosphorylation of the receiver domain modulates the activity of the output domain, The mechanisms by which receiv er domains control the activities of their respective output domains are un known, To address this question for the PhoB protein from Escherichia coli, we have employed two separate genetic approaches, deletion analysis and do main swapping. In-frame deletions were generated within the phoB gene, and the phenotypes of the mutants were analyzed. The output domain, by itself, retained significant ability to activate transcription of the phoA gene. Ho wever, another deletion mutant that contained the C terminal alpha -helix o f the receiver domain (alpha5) in addition to the entire output domain was unable to activate transcription of phoA. This result suggests that the alp ha5 helix of the receiver domain interacts with and inhibits the output dom ain. We also constructed two chimeric proteins that join various parts of t he chemotaxis response regulator, CheY, to PhoB. A chimera that joins the N -terminal similar to 85% of CheY's receiver domain to the beta5-alpha5 loop of PhoB's receiver domain displayed phosphorylation-dependent activity. Th e results from both sets of experiments suggest that the regulation of PhoB involves the phosphorylation-mediated modulation of inhibitory contacts be tween the alpha5 helix of its unphosphorylated receiver domain and its outp ut domain.