Abstract
Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63-86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63-75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic alpha-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.
Highlights
Biological membranes are subject to constant remodeling, and the control of membrane shape and curvature is essential for many vital cellular functions, such as cell division and motility, endocytosis, and vesicular trafficking [1]
Using site-directed spin labeling and EPR spectroscopy, we have shown that the endophilin N terminus undergoes a structural reorganization from an unfolded state in solution to an amphipathic helix that inserts into the membrane at the level of the head group [5]
Our first goal was to test whether the overall structure of the endophilin BAR domain is retained upon membrane interaction and, if so, how its concave surface interacts with the membrane
Summary
A functional role for this region has recently been supported by genetic rescue experiments in Drosophila [18]. We investigated the roles of helix insertion and scaffolding by examining the structure of rat endophilin A1 upon vesiculation. Previous work on annexins showed that curvaturedependent membrane interactions can lead to major conformational reorganization of a protein [19]. Our first goal was to test whether the overall structure of the endophilin BAR domain is retained upon membrane interaction and, if so, how its concave surface interacts with the membrane. We investigated the structure of the central insert (residues 64 – 86) in the membrane-bound protein using site-directed spin labeling and EPR spectroscopy. The information obtained was used in a computational refinement to generate three-dimensional atomistic models of membrane-bound endophilin
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