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：锚蛋白-1介导膜蛋白聚集和交联到 血影蛋白-肌动蛋白细胞骨架对维持红细胞的形状和机械性能至关重要 膜结合蛋白-1复合体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改变为特征的，最常见的是遗传性的 球形细胞增多症(HS)。锚蛋白介导的膜蛋白聚集的机制基础不是很好。 了解，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，如带3的阴离子交换器，并将它们与光影蛋白-肌动蛋白骨架交联，将 告诉我们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，特定成分的丢失会导致膜曲率的破坏 作为HS。我们将应对这一挑战，刻画人类的建筑、动态和规则 红细胞黏蛋白-1复合体在三个特定目的的背景下。在目标1中，我们将探讨体系结构和 组装红细胞锚蛋白-1复合体，最终目的是了解它们在体内的分布。 天然红细胞膜的背景。我们将使用单粒子低温电子显微镜来研究这一问题。 在洗涤剂胶束或脂质纳米盘中，以及施加冷冻电子，对纯化的络合物进行(低温EM) 断层摄影术(冷冻ET)和亚断层图像平均以表征结构、组成和构象 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阴离子交换器，红细胞中含量最丰富的膜蛋白，也是锚蛋白的关键结合伙伴- 1，它在跨膜气体交换中起关键作用，但其转运机制是 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- 1，并结合分子动力学，用低温电子显微镜鉴定功能相关的阴离子结合部位。 模拟。利用脂质体摄取试验鉴定重组表达突变体的功能 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负离子的调节 由磷脂酰肌醇PIP2形成的Ankyrin-1复合体。PIP2结合位点最近被 在结构上确定在带3，但该站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Ankyrin-1复合体中去除PIP2，并表征 PIP2在结构和功能上的耗竭。我们的研究将广泛影响该领域，通过解开结构 Anyrin介导的膜蛋白聚集和膜曲率调节的基础，并提供 对肌醇磷脂调节这两个过程的洞察。