CHARACTERIZATION OF NPC2, A CHOLESTEROL-BINDING PROTEIN DEFICIENT IN NIEMANN-PIC

NIEMANN-PIC 中缺乏的胆固醇结合蛋白 NPC2 的表征

• 批准号: 8170608
• 负责人: ANN M. STOCK
• 金额: $ 0.39万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170608
• 关键词: Amino Acids; Binding; Binding Sites; Cattle; Cessation of life; Cholesterol; Computer Retrieval of Information on Scientific Projects Database; Data; Disease; Epididymis; Foundations; Funding; Grant; Immunoglobulins; Inherited; Institution; Life; Ligand Binding; Mutagenesis; Proteins; Research; Research Personnel; Resolution; Resources; Source; Sterols; Structure; Supraoptic Vertical Ophthalmoplegia; Surface; United States National Institutes of Health; analog; base; cholesterol-binding protein; disulfide bond; progressive neurodegeneration; scaffold; secretory protein

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. Niemann-Pick disease type C is a fatal hereditary lysosomal storage disorder resulting in progressive neurodegeneration and death, typically in the first decade of life. The NPC2 protein, deficient in Niemann-Pick type C2 disease, is a 132-amino acid protein that was previously characterized as a major secretory protein present in mammalian epididymis and shown to function as a cholesterol transfer protein. We have solved the 1.7 angstrom resolution crystal structure of bovine NPC2. NPC2 has an immunoglobulin-like fold stabilized by three disulfide bonds. The structure reveals a loosely packed region penetrating from the surface into the hydrophobic core that forms adjacent small cavities with a total volume of ~160 cubic angstroms. We propose that this represents the incipient cholesterol-binding site that dilates to accommodate an ~740 cubic angstrom cholesterol molecule. The structure provides an important foundation for functional analysis of NPC2 and provides a scaffold for interpreting mutagenesis data. The current structure is a starting point, not an end point. It has raised many important questions. Fro example, what is the binding site for cholesterol? What conformational changes are required for sterol binding? What is the mechanism of this conformational change? Are other protein partners involved in facilitating this transition? And, can the binding pocket accommodate sterols other than cholesterol? We propose to take a structural approach to investigating these issues. We propose to use structural information as a basis for analyzing sterol-binding by NPC2. Specifically, we aim to ) determine a high resolution x-ray crystal structure of NPC2 bound to cholesterol or a closely related analog, and 2) characterize the ligand binding pocket of NPC2 using a combination of computational and experimental approaches.

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。 C型尼曼-匹克病是一种致命的遗传性溶酶体贮积症，导致进行性神经变性和死亡，通常发生在生命的第一个十年。NPC 2蛋白，在尼曼-匹克C2型疾病中缺乏，是一种132个氨基酸的蛋白，其先前被表征为哺乳动物附睾中存在的主要分泌蛋白，并显示出作为胆固醇转移蛋白的功能。 我们已经解决了牛NPC 2的1.7埃分辨率晶体结构。 NPC 2具有由三个二硫键稳定的免疫球蛋白样折叠。 该结构揭示了从表面渗透到疏水核心中的松散堆积区域，其形成总体积为~160立方埃的相邻小空腔。 我们认为，这代表了初始的胆固醇结合位点，扩张以容纳约740立方埃的胆固醇分子。 该结构为NPC 2的功能分析提供了重要基础，并为解释诱变数据提供了支架。 目前的结构是一个起点，而不是终点。 它提出了许多重要的问题。 例如，胆固醇的结合位点是什么？ 甾醇结合需要什么样的构象变化？ 这种构象变化的机制是什么？ 其他蛋白质伙伴是否参与促进这种转变？ 而且，结合口袋可以容纳胆固醇以外的固醇吗？ 我们建议采取结构性的方法来调查这些问题。 我们建议使用结构信息作为分析的基础，通过NPC 2甾醇结合。 具体而言，我们的目标是）确定与胆固醇或密切相关的类似物结合的NPC 2的高分辨率X射线晶体结构，以及2）使用计算和实验方法的组合表征NPC 2的配体结合口袋。