The Role of Conserved Residues in the DEDDh Motif: the Proton-Transfer Mechanism of HIV-1 RNase H
The Role of Conserved Residues in the DEDDh Motif: the Proton-Transfer Mechanism of HIV-1 RNase H
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DOI：
10.1021/acscatal.1c01493
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发表时间：
2021-06-16
期刊：
影响因子：
12.9
通讯作者：
Rosta, Edina
中科院分区：
文献类型：
--
作者：
Duerr, Simon L.;Bohuszewicz, Olga;Rosta, Edina
RNaseH是酶中双金属离子催化的典型例子。切割DNA/RNA杂交物的核糖核酸(RNA)骨架的RNaseH活性不仅存在于重要的药物靶点，如HIV-1逆转录酶，而且存在于许多其他裸酶中，如智人(Homo Seniens)和大肠杆菌(EC)RNase H，或者值得注意的是，在CRISPR基因编辑分子机制中的酶中。尽管它很重要，但揭示质子转移事件的反应机制尚不清楚。特别是，还不知道哪个基团是离开基团的质子给体。此外，人们提出了几种不同的质子受体，而质子受体的确切身份也是难以捉摸的。在这里，我们回顾了RNaseH的机制，其中我们发现DDE基序的高度保守的Glu残基通过糖的2‘O原子进一步稳定的机制作为质子供体。此外，我们还描述了一种替代的质子转移机制，通过同意的催化His残基来去质子化攻击的水分子。此外，我们的量子力学/分子力学(QM/MM)计算结合了哈密顿副本交换和有限温度弦方法，为HIV-1 RNaseH催化的反应提供了准确的自由能分布。我们报道的途径与突变的HIV-1、HS和EC RNaseH的动力学数据、计算的pK(A)、DEDD残基的值和结晶学研究是一致的。在磷酸盐裂解步骤中遇到的总反应势垒类似于19kcal摩尔(-1)，与类似于1-100min(-1)的缓慢实验速率相匹配。此外，利用分子动力学(MD)计算，我们对最近确定的第三个瞬时二价金属离子的结合位置进行了采样，该结合位置位于产物络合物中具有剪切性的磷酸盐附近。我们的结果解释了第三种金属离子促进产物在Aquifex aeolicus RNaseIII晶体结构中的释放和Bh RNaseH在晶体反应中的实验观察。综上所述，我们提供了一个核酸酶催化反应的分子机制，这可能是具有DDE基序的两个金属离子催化解磷酶家族的共同之处。
RNase H is a prototypical example for two-metalion catalysis in enzymes. An RNase H activity cleaving the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid is present not only in important drug targets, such as the HIV-1 reverse transcriptase, but also in many other nudeases, such as Homo sapiens (Hs) and Escherichia coil (Ec) RNase H or, notably, in enzymes that are part of the CRISPR gene editing molecular machinery. Despite its importance, the reaction mechanism uncovering the proton-transfer events is not yet understood. In particular, it is not known, which group is the proton donor for the leaving group. Moreover, several different proton acceptors were proposed, and the exact identity of the proton acceptor is also elusive. Here, we revisit the mechanism for RNAse H, whereby we find that the highly conserved Glu residue of the DDE motif acts as a proton donor via a mechanism further stabilized by the 2'O atom of the sugar. Additionally, we also describe an alternative proton-transfer mechanism via a consented catalytic His residue to deprotonate the attacking water molecule. Furthermore, our quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method provide an accurate free-energy profile for the reaction catalyzed by the HIV-1 RNase H. Our reported pathway is consistent with kinetic data obtained for mutant HIV-1, Hs, and Ec RNase H, with the calculated pK(a), values of the DEDD residues and with crystallographic studies. The overall reaction barrier of similar to 19 kcal mol(-1), encountered in the phosphate-cleavage step, matches the slow experimental rate of similar to 1-100 min(-1) Additionally, using molecular dynamics (MD) calculations, we sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Taken together, we provide a molecular mechanism of the nuclease catalytic reaction that is likely common for the broad family of twometal-ion catalytic phosphate-cleaving enzymes with a DDE motif.