PROPOSED SIGNAL-TRANSDUCTION ROLE FOR CONSERVED CHEY RESIDUE THR87, AMEMBER OF THE RESPONSE REGULATOR ACTIVE-SITE QUINTET

CheY serves as a structural prototype for the response regulator prote ins of two-component regulatory systems. Functional roles have previou sly been defined for four of the five highly conserved residues that f orm the response regulator active site, the exception being the hydrox y amino acid which corresponds to Thr87 in CheY, To investigate the co ntribution of Thr87 to signaling, we characterized, genetically and bi ochemically, several cheY mutants with amino acid substitutions at thi s position. The hydroxyl group appears to be necessary for effective c hemotaxis, as a Thr-->Ser substitution was the only one of six tested which retained a Che(+) swarm phenotype. Although nonchemotactic, cheY mutants.with amino acid substitutions T87A and T87C could generate cl ockwise flagellar rotation either in the absence of CheZ, a protein th at stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13-->Lys, demonstrating that a hydroxy am ino acid at position 87 is not essential for activation of the flagell ar switch, All purified mutant proteins examined phosphorylated effici ently from the CheA kinase in vitro but were impaired in autodephospho rylation. Thus, the mutant CheY proteins are phosphorylated to a great er degree than wild-type CheY Set support less clockwise flagellar rot ation. The data imply that Thr87 is important for generating and/or st abilizing the phosphorylation-induced conformational change in CheY. F urthermore, the various position 87 substitutions differentially affec ted several properties of the mutant proteins. The chemotaxis and auto dephosphorylation defects were tightly linked, suggesting common struc tural elements, whereas the effects on self-catalyzed and CheZ-mediate d dephosphorylation of CheY were uncorrelated, suggesting different st ructural requirements for the two dephosphorylation reactions.