Role of Dynamics and Mutations in Interactions of a Zinc Finger Antiviral Protein with CG-rich Viral RNA
Role of Dynamics and Mutations in Interactions of a Zinc Finger Antiviral Protein with CG-rich Viral RNA
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DOI：
10.1021/acs.jcim.2c01487
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发表时间：
2023-01-27
影响因子：
5.6
通讯作者：
Vashisth，Harish
中科院分区：
文献类型：
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作者：
Pal，Saikat;Kumar，Amit;Vashisth，Harish
锌指抗病毒蛋白（ZAP）是一种选择性抑制多种病毒复制的宿主抗病毒因子。ZAP识别富含CG的RNA序列并激活病毒RNA降解机制。在这项工作中，我们研究了ZAP/RNA复合物的动力学，并计算了影响ZAP与病毒RNA结合的突变的能量学。小鼠-ZAP/RNA复合物的晶体结构显示RNA与锌指2（ZF 2）和ZF 3结构域相互作用。然而，我们发现，由于单链RNA的动力学行为，RNA的末端核苷酸C1和G2从ZF 3到ZF 1结构域改变了它们的位置。此外，锌离子和病毒RNA之间的静电相互作用为ZAP/RNA复合物提供了进一步的稳定性。我们还提供了7个残基对（C1-Arg 74，C1-Arg 179，G2-Arg 74，U3-Lys 76，C4-Lys 76，G5-Arg 95和U6-Glu 204）的结构和热力学证据，这些残基对显示出有利的ZAP/RNA相互作用，尽管在ZAP/RNA晶体结构中没有观察到这些相互作用。与小鼠ZAP/RNA晶体结构的观察结果一致，我们发现四个残基对（C4-Lys 89，C4-Leu 90，C4-Tyr 108和G5-Lys 107）在MD模拟中保持稳定的相互作用。基于实验诱变研究和我们的残基水平的相互作用分析，我们选择了7个残基（Arg 74，Lys 76，Lys 89，Arg 95，Lys 107，Tyr 108和Arg 179）的个别丙氨酸突变。此外，我们还研究了仅在晶体结构中观察到的与RNA相互作用的残基（Tyr 98，Glu 148和Arg 170）中的突变。在这10个突变中，我们发现5个残基Arg 74、Lys 76、Lys 89、Lys 107和Glu 148中的每一个中的Ala突变显著降低了ZAP与RNA的结合亲和力。
Zinc finger antiviral protein (ZAP) is a host antiviral factor that selectively inhibits the replication of a variety of viruses. ZAP recognizes the CG-enriched RNA sequences and activates the viral RNA degradation machinery. In this work, we investigated the dynamics of a ZAP/RNA complex and computed the energetics of mutations in ZAP that affect its binding to the viral RNA. The crystal structure of a mouse-ZAP/RNA complex showed that RNA interacts with the zinc finger 2 (ZF2) and ZF3 domains. However, we found that due to the dynamic behavior of the single-stranded RNA, the terminal nucleotides C1 and G2 of RNA change their positions from the ZF3 to the ZF1 domain. Moreover, the electrostatic interactions between the zinc ions and the viral RNA provide further stability to the ZAP/RNA complex. We also provide structural and thermodynamic evidence for seven residue pairs (C1–Arg74, C1–Arg179, G2–Arg74, U3–Lys76, C4–Lys76, G5–Arg95, and U6–Glu204) that show favorable ZAP/RNA interactions, although these interactions were not observed in the ZAP/RNA crystal structure. Consistent with the observations from the mouse-ZAP/RNA crystal structure, we found that four residue pairs (C4–Lys89, C4–Leu90, C4–Tyr108, and G5–Lys107) maintained stable interactions in MD simulations. Based on experimental mutagenesis studies and our residue-level interaction analysis, we chose seven residues (Arg74, Lys76, Lys89, Arg95, Lys107, Tyr108, and Arg179) for individual alanine mutations. In addition, we studied mutations in those residues that are only observed in the crystal structures as interacting with RNA (Tyr98, Glu148, and Arg170). Out of these 10 mutations, we found that the Ala mutation in each of the five residues Arg74, Lys76, Lys89, Lys107, and Glu148 significantly reduced the binding affinity of ZAP to RNA.