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

摘要 肾脏的一个基本功能是恢复过滤后的水、电解质和蛋白质，以便 保存有价值的营养物质，同时丢弃最终的尿液。电解液和水的顺序回收是 然而，众所周知，人们对从滤液中捕获蛋白质的了解较少。蛋白质捕获是 由两种名为megalin和cubilin的巨大蛋白质介导。尽管这两个人的关键功能 分子，对其分子机制知之甚少，以及关于巨蛋白和 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？ 分子同时存在，还是配体之间为了结合而相互合作或竞争？这些问题 仍未得到回答的部分原因是巨蛋白和立方体蛋白的大小，600 kDa和450 kDa 这使得它们的生化、分子和结构分析都令人望而生畏。在劳动密集型的 冒险，夏皮罗和布拉什博士以及巴拉施和本肯博士已经将这些巨大的 蛋白质以及天然的巨球蛋白-丘比林-白蛋白复合体。分离出的蛋白质显示出非 聚集的、行为良好的单粒子在电子显微镜实验中的行为。3D重建自 负染色EM显示一个显著的结构，其中巨蛋白的结构域折叠形成一个大的球状结构 一种结构，其中不同大小的深裂缝和洞是由众多的巨型结构组合而成的 子域。这些缝隙和孔足够大，可以对接不同的尿液配体，如NGAL和 白蛋白。基于这些观察结果，我们认为巨蛋白可能是一块有捆绑口袋的海绵 与不同的配体互补。巨球蛋白-铜蓝蛋白-白蛋白复合体看起来更大，并具有明显的 结构特征。从这些初步数据开始，我们的目标是定义巨蛋白的结构和 超级蛋白-立方体蛋白再循环受体及其与过滤蛋白配体的复合体。我们将首先使用 单粒子冷冻-EM以指定在3D EM重建中可视化的受体亚域的身份，以及 使用这些分配来识别受体的配体相互作用区。为了实现高分辨率，一些 这些研究中将使用较小的重组受体片段进行，其结构通过 X射线结晶学。我们将通过突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化合物与SPR结合的配体 从具有明确Donnai Barrow突变的人的尿液中提取。这项工作是第一个可视化全长巨型蛋白的工作 和megalin-cubilin结构；我们期待我们的结构将连接巨型回收的功能 序列和化学的受体，我们预计这将对肾脏生物学产生变革。

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.