Structure of the constitutively active double mutant CheYD13K Y106W alone and in complex with a FliM peptide

Abstract

CheY is a member of the response regulator protein superfamily that controls the chemotactic swimming response of motile bacteria. The CheY double mutant D13K Y106W (CheY**) is resistant to phosphorylation, yet is a highly effective mimic of phosphorylated CheY in vivo and in vitro. The conformational attributes of this protein that enable it to signal in a phosphorylation-independent manner are unknown. We have solved the crystal structure of selenomethionine-substituted CheY** in the presence of its target, a peptide (FliM16) derived from the flagellar motor switch, FliM, to 1.5A resolution with an R-factor of 19.6%. The asymmetric unit contains four CheY** molecules, two with FliM16 bound, and two without. The two CheY** molecules in the asymmetric unit that are bound to FliM16 adopt a conformation similar to BeF3- -activated wild-type CheY, and also bind FliM16 in a nearly identical manner. The CheY** molecules that do not bind FliM16 are found in a conformation similar to unphosphorylated wild-type CheY, suggesting that the active phenotype of this mutant is enabled by a facile interconversion between the active and inactive conformations. Finally, we propose a ligand-binding model for CheY and CheY**, in which Ile95 changes conformation in a Tyr/Trp106-dependent manner to accommodate FliM.