Structure and Biochemical Characteristics of the Methyltransferase Domain of RNA Capping Enzyme from African Swine Fever Virus
Structure and Biochemical Characteristics of the Methyltransferase Domain of RNA Capping Enzyme from African Swine Fever Virus
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DOI：
10.1128/jvi.02029-20
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发表时间：
2021-03-01
影响因子：
5.4
通讯作者：
Zhang, Heng
中科院分区：
文献类型：
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作者：
Du, Xuejian;Gao, Zeng-Qiang;Zhang, Heng
非洲猪瘟病毒（African swine fever virus， ASFV）是一种复杂的核胞质大DNA病毒（NCLDV），是一种具有破坏性的猪疾病，迫切需要开发有效的抗ASFV疫苗和药物。mRNA的5′端旋盖过程是真核生物和许多病毒的共同特征，这种旋盖结构是mRNA稳定和高效翻译所必需的。发现ASFV蛋白pNP868R具有参与mRNA封盖的guanylytransferase （GTase）活性。在这里，我们报道了pNP868R甲基转移酶（MTase）结构域（称为pNP868R(MT)）与s -腺苷- l-蛋氨酸（AdoMet）配合物的晶体结构。构造表现为ⅰ类MTase家族特有的核心褶皱，AdoMet被束缚在一个负向的深槽中。值得注意的是，pNP868R（MT）的n端延伸是有序的，并且远离adomet结合位点，这与在天花病毒RNA capping D1亚基活性位点上的紧密构象或大多数细胞RNA capping mtase中很大程度上的无序构象不同。基于pNP868R(MT)-cap模拟复合物模型的基于结构的诱变研究揭示了参与底物识别和结合的必要残基。功能研究表明，n端延伸可能在底物识别中发挥重要作用，而不是AdoMet结合。从n端延伸到活性位点周围区域的正电荷路径为底物RNA结合和接近活性位点提供了有利的静电环境。我们的结构和生化研究为pNP868R催化的mRNA帽的甲基转移过程提供了新的见解。非洲猪瘟（ASF）是由非洲猪瘟病毒（ASFV）引起的猪高度传染性出血性病毒性疾病。到目前为止，还没有有效的药物或疫苗来预防非洲猪瘟感染。预测pNP868R蛋白负责ASFV mRNA 5'端capping过程，这对mRNA的稳定性和高效翻译至关重要。在这里，我们解决了pNP868R甲基转移酶（MTase）结构域的高分辨率晶体结构。MTase结构域结构显示典型的I类MTase家族折叠，AdoMet结合到负口袋中。基于结构的诱变研究揭示了AdoMet结合和底物RNA结合的关键和保守残基。值得注意的是，n端延伸的构象和在MTase活性中的作用与先前表征的痘病毒MTase结构域不同。我们的结构-功能研究为设计针对关键酶的潜在抗asfv抑制剂提供了基础。
African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease, and the development of effective anti-ASFV vaccines and drugs is urgently needed. The process of mRNA 5'end capping is a common characteristic in eukaryotes and many viruses, and the cap structure is required for mRNA stability and efficient translation. The ASFV protein pNP868R was found to have guanylyltransferase (GTase) activity involved in mRNA capping. Here, we report the crystal structure of the pNP868R methyltransferase (MTase) domain (termed pNP868R(MT)) in complex with S-adenosyl-L-methionine (AdoMet). The structure shows the characteristic core fold of the class I MTase family, and the AdoMet is bound in a negative, deep groove. Remarkably, the N-terminal extension of pNP868R(MT) is ordered and far away from the AdoMet-binding site, distinct from the close conformation over the active site of the poxvirus RNA capping D1 subunit or the largely disordered conformation in most cellular RNA capping MTases. Structure-based mutagenesis studies based on the pNP868R(MT)-cap analog complex model revealed essential residues involved in substrate recognition and binding. Functional studies suggest that the N-terminal extension may play an essential role in substrate recognition instead of AdoMet binding. A positively charged path stretching from the N-terminal extension to the region around the active site was suggested to provide a favorable electrostatic environment for the binding and approaching of substrate RNA to the active site. Our structure and biochemical studies provide novel insights into the methyl transfer process of the mRNA cap catalyzed by pNP868R.IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic viral disease in pigs that is caused by African swine fever virus (ASFV). There have been no effective drugs or vaccines for protection against ASFV infection until now. The protein pNP868R was predicted to be responsible for the process of mRNA 5'-end capping in ASFV, which is essential for mRNA stability and efficient translation. Here, we solved the high-resolution crystal structure of the methyltransferase (MTase) domain of pNP868R. The MTase domain structure shows a canonical class I MTase family fold, and the AdoMet binds into a negative pocket. Structure-based mutagenesis studies revealed critical and conserved residues involved in AdoMet binding and substrate RNA binding. Notably, both the conformation and the role in MTase activities of the N-terminal extension are distinct from those of the previously characterized poxvirus MTase domain. Our structure-function studies provide the basis for potential anti-ASFV inhibitor design targeting the critical enzyme.