ABSTRACT
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 Mg2+ ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties. Furthermore, substitution of one of the Mg2+-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) were replaced by various charged amino acids. Most substitutions (47 of 61) resulted in the complete loss of CheY activity, as measured by the inability to support clockwise flagellar rotation. However, 10 substitutions, all introducing a new positive charge, resulted in the loss of chemotaxis but in the retention of some clockwise flagellar rotation. Of the mutants in this set, only the previously identified CheY13DK and CheY13DR mutants displayed clockwise activity in the absence of the CheA sensor kinase. The absence of negatively charged substitution mutants with residual activity suggests that the introduction of additional negative charges into the active site is particularly deleterious for CheY function. Finally, the spatial distribution of positions at which amino acid substitutions are functionally tolerated or not tolerated is consistent with the presently accepted mechanism of response regulator activation and further suggests a possible role for Met17 in signal transduction by CheY.
Signal Transduction and Osmoregulation in Escherichia coli
