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)