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恢复过滤水，电解质和蛋白质的恢复在丢弃最终尿液的同时，可以节省有价值的营养。电解质和水的顺序恢复是然而，人们对膜酸捕获蛋白质的捕获知之甚少。蛋白质捕获是由两种称为Megalin和Cubilin的巨大蛋白质介导。尽管这两个功能的关键功能分子，对它们的分子机制知之甚少，以及有关梅加林和的基本问题 Cubilin功能仍未得到答案：单个接收器如何识别和结合如此多的不同蛋白质？什么是受体：配体化学计量和亲和力？做不同类型的蛋白质与同一受体结合分子同时，还是配体合作或互相竞争以结合？这些问题由于巨大的Megalin和Cubilin，600KDA和450KDA，尚未得到答复在实验室密集型中，它们的生化，分子和结构分析令人生畏。承诺，夏皮罗博士和布拉奇博士和巴拉斯博士和贝肯博士已经纯化为同质性蛋白质以及天然的梅加林 - 蛋白 - 钙蛋白 - 钙蛋白复合物。分离的蛋白质表现出非 - 在电子显微镜实验中，汇总，良好的单个粒子行为。 3D重建负污渍EM揭示了一个非凡的建筑，其中Megalin折叠的域形成了一个庞大的全球通过众多Megalin的关联，形成了不同大小的深缝隙和孔的结构子域。这些缝隙和孔足够大，可以隔离不同的尿配体，例如ngal和白蛋白。基于这些观察结果，我们建议梅加林可能充当带有约束口袋的“海绵” 完全到不同的配体。 Megalin-cubilin-Albumin复合物看起来更大，并且具有独特的结构特征。从这些初步数据开始，我们的目标是定义Megalin和 Megalin-蛋白蛋白蛋白质复合受体及其与过滤蛋白配体的复合物。我们将首先使用单个粒子冷冻EM分配了3D EM重建中可视化的受体子域的身份，并且使用这些分配来识别接收器的配体相互作用区域。为了实现高分辨率，有些这些研究将使用较小的重组受体片段进行，并通过结构确定 X射线晶体学。我们将通过诱变和分析评估不同受体结构域的功能 SPR的配体结合，使用已知的Megalin和Cubilin配体以及新颖的候选者隔离来自具有定义的Donnai Barrow突变的人类尿液。这项工作是第一个可视化全长梅加林的作品和Megalin-cubilin结构；我们希望我们的结构将连接巨型回收的功能序列和化学的受体，我们预计这对于肾脏生物学将是变化的。