Architecture, dynamics and regulation of erythrocyte ankyrin-1 complexes

红细胞ankyrin-1复合物的结构、动力学和调节

• 批准号: 10638440
• 负责人: Oliver Biggs Clarke
• 金额: $ 68.21万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-20 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638440
• 关键词: ANK1 gene; AQP1 gene; Actins; Address; Ammonia; Anemia; Anions; Ankyrins; Architecture; Bicarbonates; Binding; Binding Sites; Biological Assay; CD47 gene; Cell membrane; Cells; Complement; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Cytoskeleton; Data; Detergents; Diffusion; Digitonin; Erythrocyte Membrane; Erythrocytes; Event; GYPA gene; Gases; Glycophorin B; Goals; Hereditary Spherocytosis; Heterogeneity; Human; In Situ; Inherited; Lipid Bilayers; Lipids; Liposomes; Literature; Mediating; Membrane; Membrane Proteins; Methods; Micelles; Molecular Conformation; Morphologic artifacts; Mutation; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Physiological; Play; Population; Preparation; Process; Property; Proteins; Recombinants; Regulation; Reporting; Research; Resolution; Rhesus; Sampling; Scaffolding Protein; Shapes; Site; Skeleton; Sodium Channel; Spectrin; Structure; Tissues; Transmembrane Domain; Vesicle; Voltage-Gated Potassium Channel; Western Blotting; Work; conformational conversion; crosslink; dimer; experimental study; insight; mechanical properties; molecular dynamics; molecular scale; mutant; nanodisk; novel; particle; polypeptide; protein complex; protein purification; reconstruction; uptake

PROJECT SUMMARY/ABSTRACT: Ankyrin-1-mediated membrane protein clustering and crosslinking to the spectrin-actin cytoskeleton is critical to maintaining the shape and mechanical properties of the erythrocyte membrane, and disruption of the ankyrin-1 complex is a proximal cause of several hereditary anemias characterized by alterations in erythrocyte shape and stability, the most frequent of which is hereditary spherocytosis (HS). The mechanistic basis of ankyrin-mediated membrane protein clustering is not well understood, in erythrocytes or any other tissue. Understanding how ankyrin-1 clusters functionally important membrane proteins such as the band 3 anion exchanger and crosslinks them to the spectrin-actin skeleton, will inform our understanding of how ankyrins modulate membrane curvature in a physiological complex, and how loss of specific components leads to disruption of membrane curvature in the context of inherited anemias such as HS. We will approach this challenge of characterizing the architecture, dynamics and regulation of the human erythrocyte ankyrin-1 complex in the context of three specific Aims. In Aim 1, we will probe the architecture and assembly of erythrocyte ankyrin-1 complexes, with the ultimate goal of understanding their disposition in the context of native erythrocyte membranes. We will approach this using single particle cryoelectron microscopy (cryoEM) of purified complexes, in either detergent micelles or lipid nanodiscs, as well as applying cryoelectron tomography (cryoET) and sub-tomogram averaging to characterize the structure, composition and conformation of the complex in the context of native erythrocyte membrane vesicles. In Aim 2, we shift our focus to the band 3 anion exchanger, the most abundant membrane protein in the erythrocyte and a key binding partner of ankyrin- 1, which plays a key role in gas exchange across the membrane, but for which the transport mechanism is unclear. We will characterize the inward-facing state of the transporter, both alone and in complex with ankyrin- 1, and identify functionally relevant anion binding sites using cryoEM in conjunction with molecular dynamics simulations. Functional characterization of recombinantly expressed mutants using liposome uptake assays will be used to validate identified anion binding sites. In Aim 3, we will investigate regulation of the band 3 anion exchanger, and the ankyrin-1 complex, by the phosphoinositide PIP2. A PIP2 binding site has recently been structurally identified in band 3, but the functional significance of this site is unclear. We will address this by depleting PIP2 from both purified band 3, and the purified ankyrin-1 complex, and characterizing the effects of PIP2 depletion on structure and function. Our research will broadly impact the field, by unraveling the structural basis of ankyrin-mediated membrane protein clustering and modulation of membrane curvature, and provide insights into the regulation of both processes by phosphoinositides.

项目摘要/摘要：Ankyrin-1 介导的膜蛋白聚类和交联 血影蛋白-肌动蛋白细胞骨架对于维持红细胞的形状和机械特性至关重要 膜，ankyrin-1 复合物的破坏是多种遗传性贫血的近端原因 其特征是红细胞形状和稳定性的改变，其中最常见的是遗传性的 球形红细胞增多症（HS）。锚蛋白介导的膜蛋白聚集的机制基础尚不明确 可以理解，在红细胞或任何其他组织中。了解 ankyrin-1 簇在功能上的重要性 膜蛋白（例如带 3 阴离子交换剂）并将其与血影蛋白-肌动蛋白骨架交联，将 让我们了解锚蛋白如何调节生理复合体中的膜曲率，以及如何 在遗传性贫血的情况下，特定成分的损失会导致膜曲率破坏，例如 作为HS。我们将应对这一挑战，描述人类的结构、动态和调节 红细胞锚蛋白-1 复合物在三个具体目标的背景下。在目标 1 中，我们将探讨架构和 红细胞锚蛋白-1复合物的组装，最终目标是了解它们在红细胞中的分布 天然红细胞膜的背景。我们将使用单粒子冷冻电子显微镜来解决这个问题 （冷冻电镜）纯化复合物，无论是洗涤剂胶束还是脂质纳米盘，以及应用冷冻电子 断层扫描 (cryoET) 和亚断层扫描平均来表征结构、成分和构象 该复合物在天然红细胞膜囊泡的背景下。在目标 2 中，我们将注意力转移到乐队上 3 阴离子交换剂，红细胞中最丰富的膜蛋白，也是锚蛋白的关键结合伴侣 1，它在跨膜气体交换中起着关键作用，但其传输机制是 不清楚。我们将描述转运蛋白的面向内的状态，无论是单独的还是与锚蛋白复合的 1，并使用冷冻电镜结合分子动力学识别功能相关的阴离子结合位点 模拟。使用脂质体摄取测定重组表达突变体的功能表征将 用于验证已识别的阴离子结合位点。在目标 3 中，我们将研究带 3 阴离子的调节 交换器和锚蛋白-1 复合物，由磷酸肌醇 PIP2 形成。最近发现了一个 PIP2 结合位点 结构上已在带 3 中确定，但该位点的功能意义尚不清楚。我们将通过以下方式解决这个问题 从纯化的带 3 和纯化的锚蛋白-1 复合物中耗尽 PIP2，并表征 PIP2 的结构和功能耗尽。我们的研究将通过阐明结构性问题来广泛影响该领域 锚蛋白介导的膜蛋白聚类和膜曲率调节的基础，并提供 深入了解磷酸肌醇对这两个过程的调节。