Plants and microorganisms use two-component signal transduction systems (TCSs) to mediate responses to environmental stimuli. TCSs mediate responses through phosphotransfer from a conserved histidine on a sensor kinase to a conserved aspartate on the receiver domain of a response regulator. Typically, signal termination occurs through dephosphorylation of the receiver domain, which can catalyze its own dephosphorylation. Despite strong structural conservation between receiver domains, reported autodephosphorylation rate constants (k dephos ) span a millionfold range. Variable receiver domain active-site residues D ؉ 2 and T ؉ 2 (two amino acids C terminal to conserved phosphorylation site and Thr/Ser, respectively) influence k dephos values, but the extent and mechanism of influence are unclear. We used sequence analysis of a large database of naturally occurring receiver domains to design mutant receiver domains for experimental analysis of autodephosphorylation kinetics. When combined with previous analyses, k dephos values were obtained for CheY variants that contained D ؉ 2/T ؉ 2 pairs found in 54% of receiver domain sequences. Tested pairs of amino acids at D ؉ 2/T ؉ 2 generally had similar effects on k dephos in CheY, PhoB N , or Spo0F. Acid or amide residues at D ؉ 2/T ؉ 2 enhanced k dephos . CheY variants altered at D ؉ 2/T ؉ 2 exhibited rate constants for autophosphorylation with phosphoramidates and autodephosphorylation that were inversely correlated, suggesting that D ؉ 2/T ؉ 2 residues interact with aspects of the ground or transition states that differ between the two reactions. k dephos of CheY variants altered at D ؉ 2/T ؉ 2 correlated significantly with k dephos of wild-type receiver domains containing the same D ؉ 2/T ؉ 2 pair. Additionally, particular D ؉ 2/T ؉ 2 pairs were enriched in different response regulator subfamilies, suggesting functional significance. T wo-component systems (TCSs) are a prevalent means of signal transduction used by plants and microorganisms to mediate responses to stimuli (1). TCSs are present in more than 95% of sequenced bacterial genomes (2, 3), and one species can contain tens to more than a hundred TCSs. TCSs regulate a wide range of processes from cell development to virulence. Signal transduction by TCSs occurs through the transfer of phosphoryl groups between histidyl and aspartyl residues of different protein components (4). Canonically, the sensory component (the sensor kinase) is a phosphodonor to the response regulator (the response-mediating component) (4). The conserved domain in the response regulator, the receiver domain, functions as a molecular switch. The phosphorylation status of a conserved Asp on the receiver domain corresponds to turning the output response on and off. Typically, receiver domain phosphorylation initiates the output response, and dephosphorylation terminates the response. Dephosphorylation can occur with the assistance of another protein, such as a phosphatase, or by self-catalysis by the receiver domain, which is term...

No abstract