Glutamate 52-β at the α/β subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer
Glutamate 52-β at the α/β subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer
复制标题
大肠杆菌 Ia 类核糖核苷酸还原酶 α/β 亚基界面上的谷氨酸 52-β 对于自由基转移的构象门控至关重要
DOI：
10.1074/jbc.m117.783092
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发表时间：
2017-06-02
影响因子：
4.8
通讯作者：
Stubbe, JoAnne
中科院分区：
文献类型：
--
作者：
Lin, Qinghui;Parker, Mackenzie J.;Stubbe, JoAnne
核糖核苷酸还原酶（RNRs）催化核苷二磷酸底物（S）转化为脱氧核苷酸，变构效应物（e）控制它们的相对比例和量，这对DNA复制和修复的保真度至关重要。大肠杆菌Ia类RNR由α和β亚基组成，形成瞬时活性α 2 β 2复合物。急诊大肠杆菌RNR受S/e依赖性构象变化的速率限制，S/e依赖性构象变化通过跨越亚基（α/β）界面的35埃途径触发自由基起始步骤。弱的亚基亲和力和复杂的核苷酸依赖性四级结构阻碍了对RNR机制的动力学门控机制的分子理解。利用从α和β的X射线结构和来自E. coli IaRNR（Iag）中，我们鉴定并研究了α/β界面处的四个残基（β 2中的Glu（350）和Glu（52）以及α 2中的Arg（329）和Arg（639）），它们在动力学门控中具有潜在的兴趣。每个残基的突变导致活性丧失，并且除了E52 Q-β 2之外，亚基亲和力减弱。将具有2，3，5-三氟酪氨酸自由基（F3 Y122中心点）替换WT-β 2中的稳定Tyr 122（中心点）的RNR突变体（部分克服构象门控的突变）置于E52 Q背景中。将该双突变体与His（6）-α 2/S/e孵育产生能够催化途径-自由基形成（Tyr（356中心点）-β 2）的RNR，0.5当量的dCDP/F3 Y122中心点，以及在下拉测定中可分离的α 2 β 2复合物的形成2小时。S/e（GDP/TTP）阴性染色EM图像显示形成的α 2 β 2复合物的均匀性。
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of alpha and beta subunits that form a transient, active alpha 2 beta 2 complex. The E. coli RNR is rate- limited by S/e- dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 angstrom across the subunit (alpha/beta) interface. The weak subunit affinity and complex nucleotide- dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism( s) of the RNR machinery. Using a docking model of alpha 2 beta 2 created from X- ray structures of alpha and beta and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the alpha/beta interface (Glu(350) and Glu(52) in beta 2 and Arg(329) and Arg(639) in alpha 2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-beta 2, weakened subunit affinity. An RNR mutant with 2,3,5- trifluorotyrosine radical (F3Y122 center dot) replacing the stable Tyr122(center dot) in WT-beta 2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His(6)-alpha 2/S/e resulted in anRNRcapable of catalyzing pathway- radical formation (Tyr(356 center dot) -beta 2), 0.5 eq of dCDP/F3Y122 center dot, and formation of an alpha 2 beta 2 complex that is isolable in pulldown assays over 2 h. Negative stainEMimages with S/e (GDP/TTP) revealed the uniformity of the alpha 2 beta 2 complex formed.