Matching biochemical reaction kinetics to the timescales of life: structural determinants that influence the autodephosphorylation rate of response regulator proteins.
Matching biochemical reaction kinetics to the timescales of life: structural determinants that influence the autodephosphorylation rate of response regulator proteins.
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DOI：
10.1016/j.jmb.2009.07.064
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发表时间：
2009-10-09
影响因子：
5.6
通讯作者：
Silversmith RE
中科院分区：
文献类型：
--
作者：
Pazy Y;Wollish AC;Thomas SA;Miller PJ;Collins EJ;Bourret RB;Silversmith RE
在双组分调节系统中，共价磷酸化通常会激活反应调节信号蛋白，而磷酰基的水解会重新建立非活性状态。尽管三维结构和活性位点特征高度保守，但不同反应调节剂的催化自去磷酸化速率相差近 106 倍。之前的研究发现了两个可变活性位点残基，对应于大肠杆菌 CheY 残基 59 和 89，它们将反应调节剂自去磷酸化率调节约 100 倍。在这里，一组五个 CheY 突变体在对应于 CheY 残基 14、59 和 89 的可变活性位点位置上与其他“模型”反应调节因子（ArcA、CusR、DctD、FixJ、PhoB 或 Spo0F）相匹配，在功能和结构上进行了表征，试图确定调节自去磷酸化速率的机制。正如预期的那样，CheY 突变体的自去磷酸化速率相对于野生型 CheY 降低了 6 至 40 倍，但所有自去磷酸化速度仍然比各自的模型反应调节剂快 12 至 80 倍。将五种 CheY 突变体（与磷酰基类似物 BeF3− 复合）的 X 射线晶体结构与野生型 CheY 或相应的模型响应调节结构进行比较，有力地证明了磷酰基对水分子攻击的空间阻碍是增强磷酰基稳定性的一种机制。结构数据还表明，如果两个过程耦合，则阻止响应调节剂从活性构象变为非活性构象可能会延迟自去磷酸化反应，并且“58”位上的残基可能有助于速率调节。无论蛋白质背景如何（CheY 或模型反应调节剂），位置“14”、“59”和“89”处的给定氨基酸组合都采用相似的构象，这表明残基身份的知识可能足以预测自动去磷酸化率，从而预测反应调节蛋白家族中信号反应的动力学。
In two-component regulatory systems, covalent phosphorylation typically activates the response regulator signaling protein and hydrolysis of the phosphoryl group reestablishes the inactive state. Despite highly conserved three-dimensional structures and active site features, the rates of catalytic autodephosphorylation for different response regulators vary by a factor of almost 106. Previous studies identified two variable active site residues, corresponding to Escherichia coli CheY residues 59 and 89, that modulate response regulator autodephosphorylation rates about 100-fold. Here, a set of five CheY mutants, which match other “model” response regulators (ArcA, CusR, DctD, FixJ, PhoB, or Spo0F) at variable active site positions corresponding to CheY residues 14, 59 and 89, were characterized functionally and structurally in an attempt to identify mechanisms that modulate autodephosphorylation rate. As expected, the autodephosphorylation rates of the CheY mutants were reduced six- to 40-fold relative to wild type CheY, but all still autodephosphorylated 12- to 80-fold faster than their respective model response regulators. Comparison of X-ray crystal structures of the five CheY mutants (complexed with the phosphoryl group analogue BeF3−) to wild type CheY or corresponding model response regulator structures gave strong evidence for steric obstruction of the phosphoryl group from the attacking water molecule as one mechanism to enhance phosphoryl group stability. Structural data also suggested that impeding the change of a response regulator from the active to inactive conformation might retard the autodephosphorylation reaction if the two processes are coupled, and that the residue at position '58' may contribute to rate modulation. A given combination of amino acids at positions '14', '59', and '89' adopted similar conformations regardless of protein context (CheY or model response regulator), suggesting that knowledge of residue identity may be sufficient to predict autodephosphorylation rate, and hence, the kinetics of the signaling response, in the response regulator family of proteins.
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影响因子：
3.2
作者：
Dyer, Collin M.;Dahlquist, Frederick W.
通讯作者：
Dahlquist, Frederick W.
影响因子：
3.2
作者：
Appleby, JL;Bourret, RB
通讯作者：
Bourret, RB
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.9
作者：
LUKAT, GS;STOCK, AM;STOCK, JB
通讯作者：
STOCK, JB
影响因子：
4.8
作者：
Meyer, MG;Park, S;Nixon, BT
通讯作者：
Nixon, BT