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

• 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
• 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' 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

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.

摘要 肾脏的一个基本功能是回收过滤水、电解质和蛋白质， 在丢弃最后的尿液的同时保存有价值的营养。电解质和水的顺序恢复是 然而，众所周知的是，关于从滤液中捕获蛋白质知之甚少。蛋白质捕获是 由两种巨大的蛋白质megalin和cubilin介导。尽管这两个关键功能 分子，对它们的分子机制知之甚少，关于megalin和 cubilin的功能仍然没有答案：一个单一的受体如何识别和结合这么多不同的蛋白质？ 什么是受体：配体化学计量和亲和力？不同类型的蛋白质会与同一受体结合吗 分子的同时，还是配体合作或竞争彼此结合？这些问题 部分原因是巨蛋白和cubilin的分子量很大，分别为600 kDa和450 kDa， 这使得它们的生物化学、分子和结构分析令人望而生畏。在劳动密集型 承担，博士夏皮罗和布拉什和博士巴拉什和本肯已经纯化到同质化这些巨大的 蛋白质以及天然巨蛋白-巨蛋白-白蛋白复合物。分离的蛋白质显示非- 聚集的，表现良好的单粒子行为在电子显微镜实验中。3D重建从 负染电镜显示了一个显著的结构，其中megalin的结构域折叠形成一个大的球状体， 由大量巨噬细胞联合形成的深裂缝和不同大小的洞的结构 子域。这些裂缝和孔足够大以对接不同的尿配体，如NGAL和 白蛋白。基于这些观察，我们认为巨蛋白可能是一种具有结合口袋的“海绵 与不同的配体互补。巨蛋白-cubilin-白蛋白复合物看起来更大，具有明显的 结构特征从这些初步数据开始，我们的目标是定义megalin的结构， 巨蛋白-cubilin蛋白再循环受体，以及它们与过滤蛋白配体的复合物。我们将首先使用 单粒子cryo-EM，用于分配在3D EM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。为了实现高分辨率，一些 这些研究中的一项将使用较小的重组受体片段进行，并通过 X射线晶体学我们将通过诱变和分析来评估不同受体结构域的功能 利用已知的megalin和cubilin配体以及分离的新候选物， 从具有确定的Donnai巴罗突变的人的尿液中提取。这项工作是第一个可视化全长megalin 和megalin-cubilin结构;我们希望我们的结构将连接巨大的回收功能， 受体的序列和化学，我们预计这将是肾脏生物学的变革。