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

描述（由申请方提供）：人APOBEC 3G（hA 3G）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限制中发挥作用。HIV病毒感染因子（Vif）通过与hA 3 G结合并通过多聚遍在蛋白化和蛋白酶体降解介导其破坏来克服hA 3 G活性。来自申请人实验室的最近的小角X射线散射（SAXS）和生物化学研究使得能够经验性地确定溶液中的hA 3G分子形状，其揭示了伸长的催化活性的尾对尾二聚体。这些观察结果使得能够鉴定可以表达为可溶性蛋白的hA 3G的折叠结构域，并评估其对结构和活性的贡献。SAXS分析还表明，hA 3G的二聚体可以通过短RNA链桥接以产生催化失活的寡聚体，这是hA 3G的先天核酸结合能力的结果。该提议的中心假设是（i）hA 3G的二聚体和更高级形式代表“核心”结构，其相对丰度决定细胞是否允许HIV感染，和（ii）HIV蛋白Vif必须在这些结构的背景下与hA 3G相互作用以抑制这种宿主防御因子。因此，以高分辨率测定hA 3G 7Vif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阶段的目的是：（1a）制备hA 3G 7Vif复合物及其结构域以确定结晶条件;（1b）评估完整的hA 3G与确定的核酸的结合以获得用于生物物理研究的可溶性单分散材料，和（2）通过SAXS确定Vif和/或hA 3G 7Vif复合物的分子包膜以补充晶体学研究。用于评估进展到R33相的步骤是：（i）获得hA 3G、Vif或其结构域或hA 3G 7Vif复合物的晶体，和（ii）测定hA 3G 7Vif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相的具体目的是：（3）确定目标1中单个组分或复合物的结构。如果hA 3G 7Vif晶体结构不可用，则使用来自目标2的hA 3G 7Vif SAXS包膜将单个宿主或病毒晶体结构的结构域在计算上对接/拟合在一起;和（4）使用结构信息来鉴定稳定宿主/病毒分子界面的关键氨基酸，并通过生物化学和功能终点测定来验证这些观察结果。总的来说，结果将鉴定Vif与hA 3G结合所必需的关键氨基酸。这些信息将为合理的药物设计提供一个平台，这将导致一种新型的HIV/AIDS治疗方法，以改善公共卫生。公共卫生相关性声明人类蛋白APOBEC 3G是一种天然的抗HIV防御因子，通过与HIV蛋白Vif结合而失活。该提案的目标是可视化Vif如何在原子水平上识别APOBEC 3G。通过观察宿主/病毒蛋白质-蛋白质界面的化学属性，研究人员将能够创造新的HIV/AIDS治疗方法，使APOBEC 3G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.