Investigation of APOBEC3G/Vif Interactions by SAXS and X-ray Crystallography

描述（由申请人提供）：人类APOBEC3G （hA3G）是一种有效的抗病毒宿主防御蛋白，通过胞苷脱氨酶依赖性和非依赖性机制在进入后HIV限制中起作用。HIV病毒感染因子（Vif）通过与hA3G结合并通过多泛素化和蛋白体降解介导其破坏，从而克服hA3G的活性。申请人实验室最近进行的小角度x射线散射（SAXS）和生化研究使溶液中hA3G分子形状的经验测定成为可能，其显示出一种细长的催化活性，尾对尾二聚体。这些观察结果鉴定了hA3G的折叠结构域，这些结构域可以作为可溶性蛋白表达，并评估了它们对结构和活性的贡献。SAXS分析还表明，hA3G的二聚体可以通过短RNA链桥接产生无催化活性的低聚物，这是hA3G与生俱来的核酸结合能力的结果。该提议的中心假设是：(i)二聚体和高阶形式的hA3G代表了“核心”结构，其相对丰度决定了细胞是否允许HIV感染；（ii）在这些结构的背景下，HIV蛋白Vif必须与hA3G相互作用，以使这种宿主防御因子失活。因此，以高分辨率确定hA3G7Vif复合物将提高我们通过基于知识的药物设计破坏宿主/病毒蛋白质界面的能力。R21相的目的是：（1a）制备hA3G7Vif配合物及其结构域，以确定结晶条件；（1b）评估完整的hA3G与特定核酸的结合，以获得可溶的单分散材料，用于生物物理研究；(2)通过SAXS测定Vif和/或hA3G7Vif复合物的分子包膜，以补充晶体学研究。评估进入R33期的里程碑是：(i)获得hA3G、Vif或其结构域或hA3G7Vif复合物的晶体，以及（ii）确定hA3G7Vif复合物的SAXS包膜。在R33阶段，我们将利用从R21阶段获得的知识来解决晶体结构。R33阶段的具体目的是：(3)确定Aim 1中单个组分或复合物的结构。如果hA3G7Vif晶体结构不可用，单个宿主或病毒晶体结构的结构域将使用Aim 2中的hA3G7Vif SAXS包膜进行计算对接/装配在一起；(4)利用结构信息确定稳定宿主/病毒分子界面的关键氨基酸，并通过生化和功能终点分析验证这些观察结果。总的来说，这些结果将确定Vif与hA3G结合所需的关键氨基酸。这一信息将为合理的药物设计提供一个平台，这将导致一种新的艾滋病毒/艾滋病治疗方法，以改善公众健康。人类蛋白APOBEC3G是一种天然的抗HIV防御因子，通过与HIV蛋白Vif结合而失活。本提案的目标是可视化Vif如何在原子水平上识别APOBEC3G。通过观察宿主/病毒蛋白-蛋白界面的化学特性，研究人员将能够创造出新的HIV/AIDS治疗方法，使APOBEC3G免受Vif的影响，从而促进人体自身的抗HIV防御系统。

DESCRIPTION (provided by applicant): Human APOBEC3G (hA3G) is a potent antiviral host defense protein that functions in post-entry HIV restriction via cytidine deaminase-dependent and -independent mechanisms. The HIV viral infectivity factor (Vif) overcomes hA3G activity by binding to it and mediating its destruction via polyubiquitination and proteosomal degradation. Recent small angle X-ray scattering (SAXS) and biochemical studies from the applicant's lab enabled an empirical determination of the hA3G molecular shape in solution, which revealed an elongated catalytically active, tail-to-tail dimer. These observations enabled the identification of folding domains of hA3G that could be expressed as soluble proteins and evaluated for their contribution to structure and activity. The SAXS analysis also suggested that dimers of hA3G could be bridged by short RNA strands to yield catalytically inactive oligomers, which is a consequence of hA3G's innate nucleic acid binding ability. The central hypothesis of this proposal is that (i) dimeric and higher order forms of hA3G represent `core' structures whose relative abundance determines whether cells are permissive to HIV infection, and (ii) the HIV protein Vif must interact with hA3G in the context of these structures to inactivate this host defense factor. As such, the determination of hA3G7Vif complexes at high resolution will advance our ability to disrupt the host/virus protein interface through knowledge-based drug design. The Aims for the R21 phase are: (1a) To prepare the hA3G7Vif complex & domains thereof to define crystallization conditions; (1b) To evaluate intact hA3G binding to defined nucleic acids to obtain soluble, monodisperse material for biophysical studies, and (2) To determine the molecular envelope of Vif and/or hA3G7Vif complexes by SAXS in order to complement crystallographic studies. Milestones for evaluating progression into the R33 phase are: (i) attainment of crystals of hA3G, Vif, or domains thereof or an hA3G7Vif complex, and (ii) determination of SAXS envelopes of hA3G7Vif complexes. In the R33 phase we will leverage knowledge gained from the R21 phase to solve crystal structures. The Specific Aims of the R33 phase are: (3) To determine structures of individual components or complexes from Aim 1. If hA3G7Vif crystal structures are not available, the domains of individual host or viral crystal structures will be docked/fitted together computationally using the hA3G7Vif SAXS envelope from Aim 2; and (4) To use structural information to identify key amino acids that stabilize the host/virus molecular interface, and to validate these observations by biochemical and functional endpoint assays. Collectively the results will identify key amino acids necessary for Vif binding to hA3G. This information will provide a platform for rational drug design that will lead to a novel class of HIV/AIDS therapeutics to improve public health.Public Health Relevance Statement The human protein APOBEC3G is a natural, anti-HIV defense factor that becomes inactivated through association with the HIV protein Vif. The goal of this proposal is to visualize how Vif recognizes APOBEC3G at the atomic level. By observing the chemical attributes of the host/virus protein-protein interface, researchers will be able to create novel HIV/AIDS therapeutics that shield APOBEC3G from Vif, thereby promoting the body's own anti-HIV defense system.