人源APOBEC3F蛋白CD2结构域催化DNA胞嘧啶脱氨基化抗HIV感染分子机制研究

APOBEC3F (A3F) is a member of the family of deoxycytidine deaminases APOBEC, which can convert dC residue in single-stranded DNA (ssDNA) to dU, and act as a DNA editor. APOBEC3F contains two domains, A3F-CD1 at its 5’-terminus and A3F-CD2 at its 3’-terminus. A3F-CD2 not only mediates the interactions of A3F with Vif protein of the virus, but also catalyzes cytidine deamination, induces GA-to-AA hypermutation in double-stranded DNA, impairs biological function of HIV-1 virus genes, and suppresses virus replication. The deamination site of A3F-CD2 specially locates at the cytidine at 3’-terminus of the hot spot 5’-TC-3’ in the antisense ssDNA of virus gene. However, the mechanism about how A3F-CD2 specifically recognizes the hot spot 5’-TC-3’ in ssDNA and how A3F-CD2 performs its deamination reactions on cytidine is still unclear. In the project, to avoid or to stop the catalytic deamination by A3F-CD2, and to get stable complex of A3F-Cd2 with ssDNA for structural studies, we plan to firstly design ssDNA with 5’-TT-3’, 5’-AC-3’, 5’-GC-3’, etc, key sequence motifs in ssDNA, to separate T and C in 5’-TC-3’ hotspot. Then we will try to determine the structure of A3F-CD2 in complex with ssDNA by X-ray crystallography, and confirmed the complex structure by enzymology, biochemistry and cell biology assays. At the same time, we will real-time monitor cytidine deamination catalyzed by A3F-CD2 through NMR techniques. The results from this project will provide solid evidences to explain the molecular basis of how A3F-CD2 performs its deamination reaction on virus ssDNA, revealing the structural basis of A3G’s anti-virus activities.

APOBEC3F（A3F）是胞嘧啶脱氨基化酶A3家族成员，含CD1和CD2结构域。A3F-CD2既通过与HIV病毒Vif蛋白作用介导病毒壳体化，又负责催化病毒单链DNA胞嘧啶脱氨基化，诱导G –> A超突变，使HIV基因组不能执行其功能，抑制病毒复制。A3F-CD2催化胞嘧啶脱氨基化特异性发生在DNA靶序列5’-TC-3’上，分子机制与酶催化机理没有报道。靶序列的存在，不利于得到瞬态的A3F-CD2与底物DNA稳定复合物。本项目优化DNA序列, 设计包含5’-TT-3’, 5’-AC-3’及5’-GC-3’等序列的DNA，使靶序列中T与C分开，减缓或阻止催化反应，以期得到A3F-CD2与DNA稳定复合物进行结晶，利用X-衍射单晶技术解析复合物结构，利用酶学、细胞生物学等技术验证结构合理性，阐明A3F催化脱氨基化结构基础。结合核磁共振技术实时检测酶活性，探讨A3F抗HIV病毒感染的分子机制。

本项目计划开展人源APOBEC3F蛋白CD2结构域催化DNA胞嘧啶抗HIV感染的分子机制。在项目资金资助下，开展了如下工作：（1）完成了APOBEC3F CD2与两个单链DNA复合物晶体结构，揭示了APOBEC3F-CD2特异性识别DNA中胞嘧啶的分子机制；其中DNA结合位点W209, W277, Y307, Y308, F309, W310, Y314, Y333, K358, Y359, 以及A3F二聚作用位点K352K355共同发挥识别DNA的作用。最终，我们通过结构模拟说明了A3F对DNA胞嘧啶的脱氨基化机制研究结果发表在Chin J Chem, 2018上；（2）作为拓展性工作，开展了APOBEC3F同源蛋白APOBEC3G的CD2结构域与DNA相互作用机制研究；研究结果发表在Chem -An Asian J，2019上；（3）作为拓展性工作，开展了APOBEC3F的同源蛋白APOBEC3A特异性识别DNA中胞嘧啶分子机制，发现在溶液中APOBEC3A及DNA均存在两种构象，APOBEC3A通过特异性与非特异性与DNA相互作用的模式，协同识别DNA中的胞嘧啶，相关结果已经整理，准备投稿。（4）最终，发表标注项目基金号的SCI论文10篇，申请中国专利2项。承办一次国内会议（第七届华东地区结构生物学会议暨第25届上海结构生物学合作网络会议），参加国内学术会议10次（其中化学生物学会议2次），国际学术会议4次；培养博士研究生2名，硕士研究生2名。