Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis

The chemotactic responses of bacteria such as Escherichia coli and Salmonella typhimurium are mediated by phosphorylation of the CheY protein. Phospho-CheY interacts with the flagellar motor switch to cause tumbly behavior. CheY belongs to a large family of phosphorylated response regulators that function in bacteria to control motility and regulate gene expression. Residues corresponding to Asp⁶⁷, Asp¹³, and Lys¹⁰⁹ in CheY are highly conserved among all of these proteins. The site of phosphorylation in CheY is Asp⁵⁷, and in the three-dimensional structure of CheY the Asp⁵⁷ carboxylate side chain is in close proximity to the β-carboxylate of Asp¹³ and the ε-amino of Lys¹⁰⁹. To further examine the roles of these residues in response regulator function, each has been mutated to a conservative substitution, Asn for Asp and Arg for Lys. All mutations abolished CheY function in vivo. Whereas the Asp to Asn mutations dramatically reduced levels of CheY phosphorylation, the Lys to Arg mutation had the opposite effect. The high level of phosphorylation in the Lys¹⁰⁹ mutant results from a decreased autophosphatase activity as well as a lack of phosphatase stimulation by the phosphatase activating protein, CheZ. Despite its high level of phosphorylation, the Lys¹⁰⁹ mutant protein cannot produce tumbly behavior. Thus, Lys¹⁰⁹ is required for an event subsequent to phosphorylation. We propose that an interaction between the c-amino of Lysand the phosphoryl group at Aspis essential for the conformational switch that leads to activation of CheY.