A nucleobase-binding pocket in a viral RNA-dependent RNA polymerase contributes to elongation complex stability
A nucleobase-binding pocket in a viral RNA-dependent RNA polymerase contributes to elongation complex stability
复制标题
病毒RNA依赖性RNA聚合酶中的核碱基结合口袋有助于延长复合物的稳定性
DOI：
10.1093/nar/gkz1170
复制
发表时间：
2020-02-20
影响因子：
14.9
通讯作者：
Gong, Peng
中科院分区：
文献类型：
--
作者：
Shi, Wei;Ye, Han-Qing;Gong, Peng
摘要肠病毒71型(EV71)3Dpol是一种依赖RNA的RNA聚合酶(RdRP)，在病毒基因组复制中起核心作用，是抗病毒研究的重要靶点。在这里，我们报道了EV71 3Dpoll伸长络合物(EC)的晶体结构，其分辨率为1.8？结构表明，下游RNA模板的5‘端鸟苷与一个指状区口袋相互作用，碱基被H44和R277侧链通过疏水堆积相互作用夹在中间，这些相互作用在核苷酸掺入诱导的一次晶内易位事件后仍然保持，这意味着该口袋可以通过与RNA相互作用来调节聚合酶的功能性质。当突变时，残基R277在病毒学研究中显示出对病毒增殖的影响，与残基H44具有协同效应。体外生化数据进一步表明，这两个位点的突变影响RNA结合和EC的稳定性，但不影响聚合酶催化速率(Kcat)和表观NTP亲和力(Km，NTP)。我们认为，虽然很少被结晶学捕获，但类似的表面口袋与核苷酸碱基的相互作用可能普遍存在于核酸发动机酶中，以促进它们的加工能力。并对其在抗病毒药物和疫苗开发中的潜在应用进行了讨论。
Abstract The enterovirus 71 (EV71) 3Dpol is an RNA-dependent RNA polymerase (RdRP) that plays the central role in the viral genome replication, and is an important target in antiviral studies. Here, we report a crystal structure of EV71 3Dpol elongation complex (EC) at 1.8 Å resolution. The structure reveals that the 5′-end guanosine of the downstream RNA template interacts with a fingers domain pocket, with the base sandwiched by H44 and R277 side chains through hydrophobic stacking interactions, and these interactions are still maintained after one in-crystal translocation event induced by nucleotide incorporation, implying that the pocket could regulate the functional properties of the polymerase by interacting with RNA. When mutated, residue R277 showed an impact on virus proliferation in virological studies with residue H44 having a synergistic effect. In vitro biochemical data further suggest that mutations at these two sites affect RNA binding, EC stability, but not polymerase catalytic rate (kcat) and apparent NTP affinity (KM,NTP). We propose that, although rarely captured by crystallography, similar surface pocket interaction with nucleobase may commonly exist in nucleic acid motor enzymes to facilitate their processivity. Potential applications in antiviral drug and vaccine development are also discussed.