Experimental Analysis of Functional Variation within Protein Families: Receiver Domain Autodephosphorylation Kinetics

被引:16
作者
Page, Stephani C. [1 ,3 ]
Immormino, Robert M. [2 ,4 ]
Miller, Thane H. [2 ]
Bourret, Robert B. [2 ]
机构
[1] Univ N Carolina, Dept Biochem & Biophys, Chapel Hill, NC 27514 USA
[2] Univ N Carolina, Dept Microbiol & Immunol, Chapel Hill, NC USA
[3] Univ N Carolina, Dept Pharmacol, Chapel Hill, NC 27514 USA
[4] Univ N Carolina, Ctr Environm Med Asthma & Lung Biol, Chapel Hill, NC USA
关键词
AUTOPHOSPHORYLATION; PHOSPHORELAY; SEQUENCE; GENOMICS; ORDERS;
D O I
10.1128/JB.00853-15
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
引用
收藏
页码:2483 / 2493
页数:11
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