Quantifying the Relationship between Conformational Dynamics and Enzymatic Activity in Ribonuclease HI Homologues.
Quantifying the Relationship between Conformational Dynamics and Enzymatic Activity in Ribonuclease HI Homologues.
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DOI：
10.1021/acs.biochem.0c00500
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发表时间：
2020-09-08
期刊：
影响因子：
2.9
通讯作者：
Palmer AG 3rd
中科院分区：
文献类型：
--
作者：
Martin JA;Robustelli P;Palmer AG 3rd
核糖核酸酶HI （RNHI）是一种普遍存在的非序列特异性内切酶，在RNA/DNA杂交体中切割RNA链。RNHI在复制、基因组维持和逆转录病毒逆转录酶中具有必要的核糖核酸酶H结构域的功能。核磁共振波谱结合分子动力学（MD）模拟表明，大肠杆菌RNHI的扩展处理区域域在300 K时处于（底物结合能力强）“开放”和（底物结合能力弱）“封闭”状态，而嗜热热杆菌RNHI主要处于封闭状态（Stafford， K. a ., Robustelli， P., and Palmer, a . G. PLoS Computational Biology 2013, 9, 1-10）。此外，一个由计算机设计的突变型大肠杆菌Val98Ala RNHI预计主要以封闭状态填充。目前的工作验证了这一模型，并证实了所设计突变体的预测特性。MD模拟表明，手柄区域的构象偏好与Trp85、Thr92和Val101的构象相关。核磁共振残余偶极偶联常数、三键标量偶联常数和化学位移实验定义了这些残基的构象状态，从而确定了柄域的构象状态。这些核磁共振参数与RNHI同源物的Michaelis常数相关，证实了柄区在调节底物识别中的重要作用，并说明了核磁共振波谱在剖析酶功能基础上的构象偏好方面的力量。
Ribonuclease HI (RNHI), a ubiquitous, non-sequence-specific endonuclease, cleaves the RNA strand in RNA/DNA hybrids. RNHI functions in replication, genome maintenance, and retroviral reverse transcriptases contain an essential ribonuclease H domain. NMR spectroscopy combined with molecular dynamics (MD) simulations suggest a model in which the extended handle region domain of Escherichia coli RNHI populates (substrate-binding competent) “open” and (substrate-binding incompetent) “closed” states, while the thermophilic Thermus thermophilus RNHI mainly populates the closed state at 300 K (Stafford, K. A., Robustelli, P., and Palmer, A. G. PLoS Computational Biology 2013, 9, 1–10). In addition, an in silico designed mutant E. coli Val98Ala RNHI was predicted to populate primarily the closed state. The present work validates this model and confirms the predicted properties of the designed mutant. MD simulations suggest that the conformational preferences of the handle region correlate with the conformations of Trp85, Thr92, and Val101. NMR residual dipolar coupling constants, three-bond scalar coupling constants, and chemical shifts experimentally define the conformational states of these residues and hence of the handle domain. These NMR parameters correlate with the Michaelis constants for RNHI homologues, confirming the important role of the handle region in modulation of substrate recognition and illustrating the power of NMR spectroscopy in dissecting the conformational preferences underlying enzyme function.
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DOI：
10.1073/pnas.96.24.13674
发表时间：
1999-11-23
影响因子：
11.1
作者：
Hollien, J;Marqusee, S
通讯作者：
Marqusee, S
影响因子：
2.2
作者：
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通讯作者：
Bax, A
DOI：
10.1111/j.1742-4658.2009.06909.x
发表时间：
2009-03
期刊：
The FEBS journal
影响因子：
--
作者：
Champoux JJ;Schultz SJ
通讯作者：
Schultz SJ
DOI：
10.1126/science.aah3717
发表时间：
2017-01-20
期刊：
Science (New York, N.Y.)
影响因子：
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作者：
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通讯作者：
Kern D
影响因子：
16
作者：
Nowotny, Marcin;Gaidamakov, Sergei A.;Yang, Wei
通讯作者：
Yang, Wei