Structure of the Constitutively Active Double Mutant CheYD13K Y106W Alone and in Complex with a FliM Peptide

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 (FliM 16 ) derived from the flagellar motor switch, FliM, to 1.5 A resolution with an R -factor of 19.6%. The asymmetric unit contains four CheY ** molecules, two with FliM 16 bound, and two without. The two CheY ** molecules in the asymmetric unit that are bound to FliM 16 adopt a conformation similar to BeF 3 − -activated wild-type CheY, and also bind FliM 16 in a nearly identical manner. The CheY ** molecules that do not bind FliM 16 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.