SOLUTION X-RAY SCATTERING STUDIES ON MVB PATHWAY COMPONENTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 HIV颗粒通过细胞MVB途径的招募机制从宿主细胞发芽，该途径通常用于将小泡发芽进入晚期的内胚体间隔。我们建议研究MVB途径中在HIV发芽中发挥作用的三个细胞蛋白质组分：(1)Vps4，(2)Alix和(3)ESCRT I。在所有情况下，都有一些晶体结构，SAXS有望进一步了解功能上重要的组装状态和构象变化。Vps4是一种AAA-ATPase，是MVB途径的唯一酶。我们将使用SAXS建立功能相关齐聚状态的模型，确定底物结合N-末端结构域的配置，并确定其与功能相关结合伙伴Vta1相互作用的结构基础。对Alix的SAXS分析应该揭示该支架蛋白的N-结构域和V-结构域是如何组织的，并测试V-结构域结构的两个手臂随着配体结合而移动的说法。对于人ESCRT-I，我们的目标是确定组成结构域/亚单位的总体组织，并测试配体结合通过构象变化诱导自抑制的提议。我们的初步数据包括人VPS4B和酵母Vps4在Alix和ESCRT I的单体构象和结构域中的晶体结构。蛋白质和复合体已经通过定型层析和分析超速离心法进行了表征。快速获取SAXS数据证明了研究Vps4的可行性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. HIV particles bud from host cells by recruiting machinery of the cellular MVB pathway, which normally serves to bud vesicles into late endosomeal compartments. We propose to study three cellular protein components of the MVB pathway that function in HIV budding: (1) Vps4, (2) ALIX, and (3) ESCRT I. In all cases some crystal structures are available, and SAXS is expected to further understanding of functionally important assembly states and conformational changes. Vps4 is an AAA-ATPase and is the only enzyme of the MVB pathway. We will use SAXS to build a model for the functionally relevant oligomerization state, determine the disposition of the substrate-binding N-terminal domains, and determine the structural basis for its interaction with its functionally relevant binding partner Vta1. SAXS analysis of ALIX should reveal how the N-and V domains of this scaffolding protein are organized and test the proposal that the two arms of the V-domain structure move in response to ligand binding. For human ESCRT-I, we aim to determine the overall organization of component domains/subunits and test the proposal that ligand binding induces deautorepression via a conformational change. Our preliminary data include crystal structures of human VPS4B and yeast Vps4 in monomeric conformations and domains of ALIX and ESCRT I. Proteins and complexes have been characterized by sizing chromatography and analytical ultracentrifugation. Rapid access SAXS data have demonstrated the feasibility of studies on Vps4.