SOLUTION X-RAY SCATTERING STUDIES ON MVB PATHWAY COMPONENTS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 HIV颗粒通过细胞MVB途径的募集机制从宿主细胞出芽，该途径通常用于将囊泡出芽到晚期内体隔室中。我们建议研究MVB途径中在HIV出芽中起作用的三种细胞蛋白组分：（1）Vps 4，（2）阿利克斯和（3）ESCRT I。在所有情况下，一些晶体结构是可用的，小角X射线散射有望进一步了解功能上重要的组装状态和构象变化。Vps 4是一种AAA-ATP酶，是MVB途径中唯一的酶。我们将使用SAXS建立一个模型的功能相关的寡聚化状态，确定基板结合N-末端结构域的处置，并确定其与其功能相关的结合伙伴Vta 1的相互作用的结构基础。SAXS分析阿利克斯应揭示如何N和V结构域的这种支架蛋白的组织和测试的建议，两个手臂的V结构域结构移动响应配体结合。对于人ESCRT-I，我们的目标是确定组成结构域/亚基的整体组织，并测试配体结合通过构象变化诱导去自动抑制的提议。我们的初步数据包括晶体结构的人VPS 4 B和酵母VPS 4的单体构象和结构域的阿利克斯和ESCRT I。蛋白质和复合物的特征在于尺寸色谱和分析超离心。快速获取的SAXS数据证明了对Vps 4进行研究的可行性。

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.