Structural Basis of Membrane Bending by the N-BAR Protein Endophilin

Abstract

Functioning as key players in cellular regulation of membrane curvature, BAR-domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface, and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic, and that the N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin's SH3 domains, suggesting that the spatial presentation of SH3-domains rather than affinity governs the recruitment of downstream interaction partners.

Linear aggregation of proteins on the membrane as a prelude to membrane remodeling

“…Contrary to the prevailing picture of BARs on membranes, in our simulations the proteins initially aggregate in the areas of negative Gaussian curvature, i.e., in troughs. The crescent shape (28) of BARs and the way they coat tubules (14) are what has motivated the perception of them populating positively curved membrane regions. Although single N-BAR proteins were observed in the present simulations to readily explore convex areas of the membrane, it appears that the quasi-linear aggregates have very different curvature proclivities at early stages of membrane remodeling.…”

“…1073/pnas.1309819110/-/DCSupplemental. a single protein or dense N-BAR lattices were studied theoretically and experimentally (14,(23)(24)(25)(26), simulating the N-BAR protein behavior at low and intermediate surface densities provides microscopic insights into the transient structures during the membrane restructuring at physiologically relevant concentrations. This is especially important in light of recent experimental work revealing the difference in mechanics of membrane bending under low-and high-density regimes of N-BAR proteins (27) and showed that N-BAR's mode of action needs to be studied with precise control of the bound protein density.…”

“…It is becoming increasingly apparent that these proteins do more than simply imprint their shape on the membrane surface; rather, membrane remodeling is a result of a collective effort among many proteins. Recent cryo-electron microscopic reconstruction revealed a high structural organization among BARs on membrane tubules under very high protein surface densities (14), and showed a required lateral alignment of amphipathic helices for the stabilization of membrane tubes. At the same time, fluorescence experiments suggest protein crowding also may be responsible for membrane remodeling resulting in the same membrane structures as with the binding of N-BAR domains (15).…”

“…Protein-induced membrane remodeling is an intriguing problem from both structural and biophysical standpoints, so a computational approach connecting the underlying physics to the observed behavior may help reveal a more complete scope of the mechanism. Our recently developed coarse-grained (CG) models of N-BAR domain proteins (14,18,19) and of lipid bilayers (20) form the basis for the present work. The forces between CG lipids are derived from their underlying atomic interactions, using the multiscale coarse-graining method (21,22), which minimizes the number of adjustable ad hoc parameters.…”

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…Generating curvature in a phospholipid bilayer implies that some form of packing asymmetry exists between the two leaflets of the membrane (Kirchhausen 2012). In principle, there are at least four ways in which cytosolic EAPs may contribute to lipid asymmetry and membrane curvature during CCP invagination: (1) by locally clustering lipids with larger-than-average head groups through interaction with membrane-associated proteins (Zimmerberg and Kozlov 2006), by converting one lipid type to another (Kooijman et al 2003) or by actively transporting a lipid type(s) from one leaflet to the other (Rauch and Farge 2000;Roelants et al 2010); (2) by "floating" an amphipathic helix in the inner leaflet of the membrane (Ford et al 2002) or by inserting a hydrophobic loop into the inner leaflet of the membrane (Plomann et al 2010); (3) by association of a precurved membrane-binding protein domain (such as BAR, F-BAR, or i-BAR) with the membrane surface (Mim et al 2012); or, most simply, (4) by membrane-associated protein crowding (Stachowiak et al 2012). The different mechanisms for generating membrane curvature are summarized in Figure 2.…”