Alteration of a nonconserved active site residue in the chemotaxis response regulator CheY affects phosphorylation and interaction with CheZ

CheY is a response regulator in the well studied two-component system that mediates bacterial chemotaxis, Phosphorylation of CheY at Asp(57) enhances its interaction with the flagellar motor. Asn(59) is located near the phosphorylation site, and possible roles this residue may play in CheY function were explored by mutagenesis. Cells containing CheY59NR or CheY59NH exhibited hyperactive phenotypes (clockwise flagellar rotation), and CheY59NR was characterized biochemically, A continuous enzyme-linked spectroscopic assay that monitors Pi concentration was the primary method for kinetic analysis of phosphorylation and dephosphorylation, CheY59NR autodephosphorylated at the same rate as wild-type CheY and phosphorylated similarly to wild type with acetyl phosphate and faster (4-14x) with phosphoramidate and monophosphoimidazole. CheY59NR was extremely resistant to CheZ, requiring at least 250 times more CheZ than wild-type CheY to achieve the same dephosphorylation rate enhancement, whereas CheY59NA was CheZ-sensitive, However, several independent approaches demonstrated that CheY59NR bound tightly to CheZ, A submicromolar Kd for CheZ binding to CheY59NR-P or CheY.BeF3- was inferred from fluorescence anisotropy measurements of fluoresceinated-CheZ. A complex between CheY59NR-P and CheZ was isolated by analytical gel filtration, and the elution position from the column was indistinguishable from that of the CheZ dimer, Therefore, we were not able to detect large CheY-P.CheZ complexes that have been inferred using other methods. Possible structural explanations for the specific inhibition of CheZ activity as a result of the arginyl substitution at CheY position 59 are discussed.