CheY is a response regulator in the well studied twocomponent 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 P i 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 -14؋) 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 K d for CheZ binding to CheY59NR-P or CheY⅐BeF 3 ؊ 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-P⅐CheZ 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.

SummaryTwo-component regulatory systems, typically composed of a sensor kinase to detect a stimulus and a response regulator to execute a response, are widely used by microorganisms for signal transduction. Response regulators exhibit a high degree of structural similarity and undergo analogous activating conformational changes upon phosphorylation. The activity of particular response regulators can be increased by specific amino acid substitutions, which either prolong the lifetime or mimic key features of the phosphorylated state. We probed the universality of response regulator activation by amino acid substitution. Thirty-six mutations that activate 11 different response regulators were identified from the literature. To determine whether the activated phenotypes would be retained in the context of a different response regulator, we recreated 51 analogous amino acid substitutions at corresponding positions of CheY. About 55% of the tested substitutions completely or partially inactivated CheY, ª ª ª ª 30% were phenotypically silent, and ª ª ª ª 15% activated CheY. Three previously uncharacterized activated CheY mutants were found. The 94NS (and presumably 94NT) substitutions resulted in resistance to CheZ-mediated dephosphorylation. The 113AP substitution led to enhanced autophosphorylation and may increase the fraction of non-phosphorylated CheY molecules that populate the activated conformation. The locations of activating substitutions on the response regulator three-dimensional structure are generally consistent with current understanding of the activation mechanism. The best candidates for potentially universal activating substitutions of response regulators identified in this study were 13DK and 113AP.

In a two-component regulatory system, an important means of signal transduction in microorganisms, a sensor kinase phosphorylates a response regulator protein on an aspartyl residue, resulting in activation. The active site of the response regulator is highly charged (containing a lysine, the phosphorylatable aspartate, two additional aspartates involved in metal binding, and an Mg 2؉ ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties. Furthermore, substitution of one of the Mg 2؉ -coordinating aspartates with lysine or arginine in the Escherichia coli chemotaxis response regulator CheY results in phosphorylation-independent activation. In order to examine the consequences of altered charge distribution for response regulator activity and to identify possible additional amino acid substitutions that result in phosphorylation-independent activation, we made 61 CheY mutants in which residues close to the site of phosphorylation (Asp57)

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