Structure and mechanism of the protein-capture receptors of the kidney proximal tubule

肾近曲小管蛋白捕获受体的结构和机制

基本信息

批准号：
10399617
负责人：
JONATHAN M. BARASCH
金额：
$ 63.14万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-15 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10399617
关键词：
3-Dimensional Affect Affinity Albumins Architecture Behavior Binding Binding Proteins Binding Sites Biochemical Biology Biophysics Bowman&apos s space CRISPR/Cas technology Cell Fractionation Charge Chemistry Chromatography Cloning Complex Cryoelectron Microscopy Data Detergents Docking Electrolytes Electron Microscopy Endocytosis Epithelial Filtration Goals Grant Hemoglobin Human Impairment Insulin Kidney LCN2 gene LDL-Receptor Related Protein 2 Length Ligand Binding Ligand Binding Domain Ligands Light-Chain Immunoglobulins Location Logic Maps Mediating Molecular Mus Mutagenesis Mutation Negative Staining Nutrient Point Mutation Porifera Process Proteins Protocols documentation Proximal Kidney Tubules Recombinants Recovery Renal function Resolution Retinol Binding Proteins Secondary to Site Site-Directed Mutagenesis Structural Models Structure Surface Testing Time Urine Validation Visualization Vitamin D-Binding Protein Water Work X-Ray Crystallography base biophysical model biophysical techniques design experimental study facio-oculo-acoustico-renal syndrome glomerular filtration in vivo intrinsic factor-cobalamin receptor loss of function mutation mouse model mutant novel particle receptor receptor recycling reconstruction renal damage stoichiometry transmission process urinary

项目摘要

Abstract A fundamental function of the kidney is the recovery of filtered water, electrolytes, and proteins in order to conserve valuable nutrients while discarding the final urine. The sequential recovery of electrolytes and water is well understood, however, less is known about the capture of proteins from the filtrate. Protein capture is mediated by two enormous proteins called megalin and cubilin. Despite the critical function of these two molecules, little is known about their molecular mechanisms, and fundamental questions about megalin and cubilin function remain unanswered: How does a single receptor recognize and bind so many different proteins? What is the receptor:ligand stoichiometry and affinity? Do different types of proteins bind to the same receptor molecule at the same time, or do ligands cooperate or compete with one another for binding? These questions have remained unanswered in part because of the large sizes of megalin and cubilin, 600kDa and 450kDa respectively, making their biochemical, molecular and structural analysis daunting. In a labor-intensive undertaking, Drs Shapiro and Brasch and Drs Barasch and Beenken have purified to homogeneity these massive proteins as well as a native megalin-cubilin-albumin complex. The isolated proteins demonstrated non- aggregated, well-behaved single particle behavior in electron microscopy experiments. 3D reconstructions from negative stain EM reveal a remarkable architecture, in which the domains of megalin fold to form a large globular structure in which deep crevices and holes of different sizes are formed by association of the numerous megalin sub-domains. These crevices and holes are large enough to dock different urinary ligands such as NGAL and albumin. Based on these observations, we propose that megalin may act as a “sponge” with binding pockets complementary to different ligands. The megalin-cubilin-albumin complex appears larger and has distinct structural features. Beginning with these preliminary data, our goal is to define the structure of the megalin and megalin-cubilin protein-recycling receptors, and their complexes with filtered-protein ligands. We will first use single particle cryo-EM to assign the identities of receptor sub-domains visualized in 3D EM reconstructions, and use these assignments to identify ligand-interacting regions of the receptors. To achieve high resolution, some of these studies will be performed with smaller recombinant receptor fragments with structure determination by x-ray crystallography. We will assess the function of different receptor domains with mutagenesis and analysis of ligand binding by SPR, using both known megalin and cubilin ligands as well as novel candidates isolated from urine of humans with defined Donnai Barrow mutations. This work is the first to visualize full-length megalin and megalin-cubilin structures; we expect that our structures will connect the function of the giant recycling receptors to sequence and chemistry, and we expect this will be transformative for kidney biology.

抽象的肾脏的基本功能是回收过滤后的水、电解质和蛋白质，以便保留宝贵的营养物质，同时丢弃最终的尿液。电解质和水的顺序回收。然而，人们对从滤液中捕获蛋白质知之甚少。尽管这两种蛋白具有重要的功能，但它们是由两种巨大的蛋白质巨蛋白和cubilin介导的。分子，对其分子机制以及关于巨蛋白和巨蛋白的基本问题知之甚少 cubilin 的功能仍未得到解答：单个受体如何识别并结合如此多不同的蛋白质？什么是受体：配体化学计量和亲和力？不同类型的蛋白质是否与同一受体结合？分子同时结合，或者配体相互合作还是竞争结合？由于巨蛋白和 cubilin 尺寸较大，分别为 600kDa 和 450kDa，该问题仍未得到解答分别使它们的生化、分子和结构分析变得令人畏惧。夏皮罗博士和布拉什博士以及巴拉什博士和宾肯博士将这些巨大的蛋白质以及天然巨蛋白-cubilin-白蛋白复合物被证明是非-的。电子显微镜实验中聚合的、表现良好的单粒子行为。负染色电镜揭示了一种非凡的结构，其中巨蛋白域折叠形成一个大的球状无数巨蛋白结合而成的深裂隙和不同大小孔洞的结构这些子结构域足够大，可以对接不同的尿配体，例如 NGAL 和基于这些观察，我们提出巨蛋白可能充当具有结合袋的“海绵”。巨蛋白-cubilin-白蛋白复合物与不同的配体互补。从这些初步数据开始，我们的目标是定义巨蛋白的结构和我们将首先使用 megalin-cubilin 蛋白质回收受体及其与过滤蛋白质配体的复合物。单粒子冷冻电镜分配 3D 电镜重建中可视化的受体子域的身份，以及使用这些分配来识别受体的配体相互作用区域以实现高分辨率。这些研究中的一部分将使用较小的重组受体片段进行，并通过以下方法确定结构：我们将通过突变和分析来评估不同受体结构域的功能。使用已知的巨蛋白和 cubilin 配体以及分离的新候选物，通过 SPR 进行配体结合这项工作是第一个将全长巨蛋白可视化的工作。和 megalin-cubilin 结构；我们希望我们的结构能够连接巨型回收的功能；受体序列和化学，我们预计这将给肾脏生物学带来变革。