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。 C型尼曼-皮克病是一种致命的遗传性溶酶体储存障碍，导致进行性神经变性和死亡，通常在生命的第一个十年。NPC2蛋白是一种由132个氨基酸组成的蛋白，是存在于哺乳动物附睾部的一种主要分泌蛋白，具有胆固醇转移蛋白的功能，在Niemann-Pick C2型疾病中缺失。我们已经解决了牛NPC2的1.7埃分辨晶体结构。NPC2有一个免疫球蛋白样折叠，由三个二硫键稳定。该结构显示了一个从表面渗透到疏水核心的松散填充区域，该区域形成了总体积约160立方埃的相邻小空穴。我们认为这代表了早期的胆固醇结合部位，它可以膨胀以容纳~740立方埃的胆固醇分子。该结构为NPC2的功能分析提供了重要的基础，并为解释突变数据提供了支架。当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，胆固醇的结合部位是什么？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？其他蛋白质合作伙伴是否参与了这一转变？而且，绑扎袋还能容纳胆固醇以外的其他类固醇吗？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NPC2与甾醇结合的基础。具体地说，我们的目标是)确定NPC2与胆固醇或密切相关类似物结合的高分辨率X射线晶体结构，以及2)使用计算和实验相结合的方法表征NPC2的配体结合口袋。