HDX and NMR Core

摘要 蛋白质是一种动态分子，它经历着不断的弯曲和运动。蛋白质构象变化 和动力学是重要的分子识别，这些变化发生在许多关键的生物学过程中， 包括酶的激活和抑制、配体与受体的结合、翻译后 修饰和跨蛋白质-蛋白质界面的变构通信。所有这些过程都是 通过功能复合物内蛋白质的结构和动力学进行调节。因此， 研究蛋白质结构和动力学对于理解生物学过程和帮助 开发用于疾病治疗干预的靶向蛋白质的策略。HDX-NMR核心将解决 HIVE中心的中心目标包括HIV蛋白相互作用的特征， 用宿主分子或小分子探针。HDX工作的一个关键重点将是结构和动力学 HIV-1逆转录酶（RT）起始复合物，或RTIC，一种大的蛋白质-核酸复合物 包含RT（p66/p51异源二聚体）、5 ′非翻译病毒RNA（vRNA）的引物结合位点区域和 人类tRNA（项目3：Arnold、Musier-Forsyth、Griffin、Lyumkis和Sarafianos）。我们亦会研究 HIV-1 RT与APOBEC 3（A3）限制因子家族成员（最显著的是A3 G）的相互作用， 据报道，其抑制RT的聚合酶活性，也将在项目3中进行研究。在 除了慢病毒HIV-1的RTIC外，我们还将研究HIV病毒原型泡沫病毒的RTIC （PFV）。我们正在采取多方面的方法来了解这些重要的病毒-宿主复合体 使用cryo-EM、X射线晶体学、HDX、SAXS和预稳态动力学分析来研究相互作用。之间 HDX解决的主要问题是：与DNA/DNA相比，vRNA/tRNA如何与RT相互作用 和DNA/RNA底物（冷冻EM、X射线、HDX、动力学）？RNA结构在这个复合体中的作用是什么 以及它如何与RT（冷冻EM，X射线，HDX）相互作用？引发的动力学性质是什么 复杂（冷冻EM，SAXS，HDX，动力学）？HIV-1和PFV RTIC中的相互作用有何不同？如何 HIV-1 RT与A3蛋白相互作用有助于HIV-1限制？HDX一直是研究 基于喹啉的变构整合酶（IN）抑制剂（ALLINI）诱导全长野生型 型（WT）IN及其应用现在将扩展到研究最近发现的（核心4）噻吩- 的抑制剂改变IN结构（项目1）。HDX-MS还将用于探测蛋白质间相互作用 在HIV-1 Gag和Gag-Pol蛋白中（项目2），耐药突变或进化枝间差异的影响 在HIV-1蛋白质的蛋白质间相互作用（项目5），以及HIV-1蛋白质和SuFEx或 其他小分子探针（项目6）。NMR将用于支持项目6和岩心4。

Abstract Proteins are dynamic molecules that undergo constant flexing and motion. Changes in protein conformation and dynamics are important to molecular recognition and these changes occur during many key biological processes including activation and inhibition of enzymes, ligand binding to receptors, posttranslational modifications, and allosteric communication across protein-protein interfaces. All of these processes are regulated through the structure and dynamics of the proteins within the functional complex. Thus, techniques to study protein structure and dynamics are critical to the understanding of biological processes and to aid in developing strategies to target proteins for therapeutic intervention in disease. The HDX-NMR Core will address central goals of the HIVE Center that include characterization of HIV protein interactions among themselves, with host molecules, or with small molecule probes. A key focus of HDX efforts will be the structure and dynamics of the HIV-1 reverse transcriptase (RT) initiation complex, or RTIC, a large protein-nucleic acid complex comprising RT (p66/p51 heterodimer), the primer-binding site region of the 5’ untranslated viral RNA (vRNA) and a human tRNA (Project 3: Arnold, Musier-Forsyth, Griffin, Lyumkis, and Sarafianos). We will also investigate the interactions of HIV-1 RT with members of the APOBEC3 (A3) family of restriction factors (most notably A3G), which have been reported to suppress the polymerase activity of RT and will also be studied in Project 3. In addition to the RTIC from lentivirus HIV-1, we will also investigate the RTIC of spumavirus prototype foamy virus (PFV). We are taking a multifaceted approach towards understanding these important virus-host complex interactions, using cryo-EM, X-ray crystallography, HDX, SAXS, and pre-steady state kinetic analyses. Among the main questions addressable by HDX are: How does the vRNA/tRNA interact with RT compared to DNA/DNA and DNA/RNA substrates (cryo-EM, X-ray, HDX, kinetics)? What is the role of RNA structure in this complex and how does it interact with RT (cryo-EM, X-ray, HDX)? What are the dynamic properties of the initiation complex (cryo-EM, SAXS, HDX, kinetics)? How do interactions in the HIV-1 and PFV RTICs differ? How does HIV-1 RT interact with A3 proteins to contribute to HIV-1 restriction? HDX has been instrumental for studying quinoline-based allosteric integrase (IN) inhibitor (ALLINIs) induced aberrant multimerization of full-length wild type (WT) IN and its application will now be extended to examine how recently discovered (Core 4) thiophene- based inhibitors alter the IN structure (Project 1). HDX-MS will also be used to probe inter-protein interactions among HIV-1 Gag and Gag-Pol proteins (Project 2), the effect of resistance mutations or inter-clade differences in inter-protein interactions in HIV-1 proteins (Project 5), and interactions between HIV-1 proteins and SuFEx or other small molecule probes (Project 6). NMR will be used to support Project 6 and Core 4.