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.

项目总结/摘要：锚蛋白-1介导的膜蛋白聚集和交联 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是至关重要的 锚蛋白-1复合物的破坏是几种遗传性贫血的近端原因 以红细胞形状和稳定性的改变为特征，其中最常见的是遗传性的 球形红细胞增多症（HS）。锚定蛋白介导的膜蛋白聚集的机制基础尚不清楚 在红细胞或任何其他组织中。了解锚蛋白-1如何在功能上重要 膜蛋白如带3阴离子交换剂，并将它们交联到血影蛋白-肌动蛋白骨架上， 告诉我们锚蛋白如何在生理复合体中调节膜曲率，以及如何 在遗传性贫血的情况下，特定成分的丢失导致膜曲率的破坏， 如HS。我们将处理这一挑战的特点架构，动态和调节的人类 红细胞锚蛋白-1复合物在三个特定目的的背景下。在目标1中，我们将探讨架构， 红细胞锚蛋白-1复合物的组装，最终目标是了解它们在细胞中的分布。 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处理这个问题 纯化的复合物，在洗涤剂胶束或脂质纳米盘，以及应用冷冻电子显微镜（cryoEM）， 断层扫描（cryoET）和亚断层图像平均，以表征结构、组成和构象 的背景下，天然红细胞膜囊泡的复合物。在目标2中，我们将重点转移到乐队 3阴离子交换剂，红细胞中最丰富的膜蛋白和锚蛋白的关键结合伴侣- 1，其在跨膜气体交换中起关键作用，但其运输机制是 不清楚我们将描述转运蛋白的内向状态，无论是单独的还是与锚蛋白复合的- 1，并使用cryoEM结合分子动力学鉴定功能相关的阴离子结合位点 模拟使用脂质体摄取测定法对重组表达的突变体进行功能表征， 用于验证已鉴定的阴离子结合位点。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，通过磷酸肌醇PIP 2。PIP 2结合位点最近被 在结构上鉴定在带3中，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一问题： 从纯化的带3和纯化的锚蛋白-1复合物中去除PIP 2，并表征 PIP 2缺失对结构和功能的影响。我们的研究将广泛影响该领域，通过解开结构 的基础上，锚定介导的膜蛋白聚类和膜曲率的调制，并提供 对磷酸肌醇调节这两个过程的见解。