ABSTRACT 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. IMPORTANCE One protein family, defined by a conserved domain, can include hundreds of thousands of known members. Characterizing conserved residues within a conserved domain can identify functions shared by all family members. However, a general strategy to assess features that differ between members of a family is lacking. Fully exploring the impact of just two variable positions within a conserved domain could require assessment of 400 (i.e., 20 × 20) variants. Instead, we created and analyzed a nonredundant database of receiver domain sequences. Five percent of D + 2/T + 2 pairs were sufficient to represent 50% of receiver domain sequences. Using protein sequence analysis to prioritize mutant choice made it experimentally feasible to extensively probe the influence of positions D + 2 and T + 2 on receiver domain autodephosphorylation kinetics
