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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：
10190932
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/10190932
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 Molecular Structure 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.

摘要