Architecture, dynamics and regulation of erythrocyte ankyrin-1 complexes

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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.