Roles of Amphipathic Helices and the Bin/Amphiphysin/Rvs (BAR) Domain of Endophilin in Membrane Curvature Generation

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 α-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.     

Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis

Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilin's SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.     

Endophilin BAR domain drives membrane curvature by two newly identified structure-based mechanisms

The crescent-shaped BAR (Bin/Amphiphysin/Rvs-homology) domain dimer is a versatile protein module that senses and generates positive membrane curvature. The BAR domain dimer of human endophilin-A1, solved at 3.1 A, has a unique structure consisting of a pair of helix-loop appendages sprouting out from the crescent. The appendage's short helices form a hydrophobic ridge, which runs across the concave surface at its center. Examining liposome binding and tubulation in vitro using purified BAR domain and its mutants indicated that the ridge penetrates into the membrane bilayer and enhances liposome tubulation. BAR domain-expressing cells exhibited marked plasma membrane tubulation in vivo. Furthermore, a swinging-arm mutant lost liposome tubulation activity yet retaining liposome binding. These data suggested that the rigid crescent dimer shape is crucial for the tubulation. We here propose that the BAR domain drives membrane curvature by coordinate action of the crescent's scaffold mechanism and the ridge's membrane insertion in addition to membrane binding via amino-terminal amphipathic helix.     

Mechanism of endophilin N-BAR domain-mediated membrane curvature

Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin-A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N-BAR domains function as low-affinity dimers regulating binding partner recruitment to areas of high membrane curvature.     

Structural basis for the actin-binding function of missing-in-metastasis

The adaptor protein missing-in-metastasis (MIM) contains independent F- and G-actin binding domains, consisting, respectively, of an N-terminal 250 aa IRSp53/MIM homology domain (IMD) and a C-terminal WASP-homology domain 2 (WH2). We determined the crystal structures of MIM's IMD and that of its WH2 bound to actin. The IMD forms a dimer, with each subunit folded as an antiparallel three-helix bundle. This fold is related to that of the BAR domain. Like the BAR domain, the IMD has been implicated in membrane binding. Yet, comparison of the structures reveals that the membrane binding surfaces of the two domains have opposite curvatures, which may determine the type of curvature of the interacting membrane. The WH2 of MIM is longer than the prototypical WH2, interacting with all four subdomains of actin. We characterize a similar WH2 at the C terminus of IRSp53 and propose that in these two proteins WH2 performs a scaffolding function.     

Crystal structures of the BAR-PH and PTB domains of human APPL1

APPL1 interacts with adiponectin receptors and other important signaling molecules. It contains a BAR and a PH domain near its N terminus, and the two domains may function as a unit (BAR-PH domain). We report here the crystal structures of the BAR-PH and PTB domains of human APPL1. The structures reveal novel features for BAR domain dimerization and for the interactions between the BAR and PH domains. The BAR domain dimer of APPL1 contains two four-helical bundles, whereas other BAR domain dimers have only three helices in each bundle. The PH domain is located at the opposite ends of the BAR domain dimer. Yeast two-hybrid assays confirm the interactions between the BAR and PH domains. Lipid binding assays show that the BAR, PH, and PTB domains can bind phospholipids. The ability of APPL1 to interact with multiple signaling molecules and phospholipids supports an important role for this adaptor in cell signaling.     

Structure and plasticity of Endophilin and Sorting Nexin 9

Endophilin and Sorting Nexin 9 (Snx9) play key roles in endocytosis by membrane curvature sensing and remodeling via their Bin/Amphiphysin/Rvs (BAR) domains. BAR and the related F-BAR domains form dimeric, crescent-shaped units that occur N- or C-terminally to other lipid-binding, adaptor, or catalytic modules. In crystal structures, the PX-BAR unit of Snx9 (Snx9(PX-BAR)) adopts an overall compact, moderately curved conformation. SAXS-based solution studies revealed an alternative, more curved state of Snx9(PX-BAR) in which the PX domains are flexibly connected to the BAR domains, providing a model for how Snx9 exhibits lipid-dependent curvature preferences. In contrast, Endophilin appears to be rigid in solution, and the SH3 domains are located at the distal tips of a BAR domain dimer with fixed curvature. We also observed tip-to-tip interactions between the BAR domains in a trigonal crystal form of Snx9(PX-BAR) reminiscent of functionally important interactions described for F-BAR domains.     

The BAR-domain family of proteins: a case of bending and binding?

BAR-domains recently took centre stage in science through a report on the crystal structure of this domain in Drosophila Amphiphysin. Though only weakly conserved at the sequence level, the structure of the BAR domain shows striking similarity to the GTPase-binding domain of Arfaptin 2, an effector of Rho- and Arf- GTPases. On the basis of this sequence and structural similarity, these two proteins have been classified as belonging to the same family, the BAR-domain family, and they probably also have similar functional characteristics. Presented here are the results of a database search for the sequence of the BAR domain of Amphiphysin and Arfaptin 2. This search identified a variety of related proteins, most of which are involved in intracellular transport and especially in endocytosis. For example, the BAR-domain family includes Endophilins, GTPase-activating proteins of the Centaurinbeta family and Oligophrenins, the adaptor proteins APPL1 and APPL2 that were recently shown to interact with the small GTPase Rab5, as well as members of the Sorting nexin family. On the basis of the structures of Amphiphysin and Arfaptin 2 and the cellular role of Amphiphysins in the early steps of endocytosis, the functions of the BAR domain have been defined as a dimerization motif and as sensing and inducing membrane curvature. However, data on Arfaptin 2 and now also on the Adaptor proteins APPL1 and 2 suggest that another function of the BAR domain is to bind to small GTPases.     

Voltage-dependent calcium channels at the plasma membrane, but not vesicular channels, couple exocytosis to endocytosis

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.     

Endophilin-1: a multifunctional protein

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.     

Dissecting BAR Domain Function in the Yeast Amphiphysins Rvs161 and Rvs167 during Endocytosis

BAR domains are protein modules that bind to membranes and promote membrane curvature. One type of BAR domain, the N-BAR domain, contains an additional N-terminal amphipathic helix, which contributes to membrane-binding and bending activities. The only known N-BAR-domain proteins in the budding yeast Saccharomyces cerevisiae, Rvs161 and Rvs167, are required for endocytosis. We have explored the mechanism of N-BAR-domain function in the endocytosis process using a combined biochemical and genetic approach. We show that the purified Rvs161–Rvs167 complex binds to liposomes in a curvature-independent manner and promotes tubule formation in vitro. Consistent with the known role of BAR domain polymerization in membrane bending, we found that Rvs167 BAR domains interact with each other at cortical actin patches in vivo. To characterize N-BAR-domain function in endocytosis, we constructed yeast strains harboring changes in conserved residues in the Rvs161 and Rvs167 N-BAR domains. In vivo analysis of the rvs endocytosis mutants suggests that Rvs proteins are initially recruited to sites of endocytosis through their membrane-binding ability. We show that inappropriate regulation of complex sphingolipid and phosphoinositide levels in the membrane can impinge on Rvs function, highlighting the relationship between membrane components and N-BAR-domain proteins in vivo.     

Bar domain proteins: a role in tubulation, scission and actin assembly in clathrin-mediated endocytosis

Endocytosis is an important way for cells to take up liquids and particles from their environment. It requires membrane bending to be coupled with membrane fission, and the actin cytoskeleton has an active role in membrane remodelling. Here, we review recent research into the function of Bin-Amphiphysin-Rvs (BAR) domain proteins, which can sense membrane curvature and recruit actin to membranes. BAR proteins interact with the endocytic and cytoskeletal machinery, including the GTPase dynamin (which mediates vesicle fission), N-WASP (an Arp2/3 complex regulator) and synaptojanin (a phosphoinositide phosphatase). We describe three classes of BAR domains, BAR, N-BAR and F-BAR, providing examples of each discussing and how they function in linking membranes to the actin cytoskeleton in endocytosis.     

Single point mutation in Bin/Amphiphysin/Rvs (BAR) sequence of endophilin impairs dimerization, membrane shaping, and Src homology 3 domain-mediated partnership

Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are essential players in the dynamics of intracellular compartments. The BAR domain is an evolutionarily conserved dimeric module characterized by a crescent-shaped structure whose intrinsic curvature, flexibility, and ability to assemble into highly ordered oligomers contribute to inducing the curvature of target membranes. Endophilins, diverging into A and B subgroups, are BAR and SH3 domain-containing proteins. They exert activities in membrane dynamic processes such as endocytosis, autophagy, mitochondrial dynamics, and permeabilization during apoptosis. Here, we report on the involvement of the third α-helix of the endophilin A BAR sequence in dimerization and identify leucine 215 as a key residue within a network of hydrophobic interactions stabilizing the entire BAR dimer interface. With the combination of N-terminal truncation retaining the high dimerization capacity of the third α-helices of endophilin A and leucine 215 substitution by aspartate (L215D), we demonstrate the essential role of BAR sequence-mediated dimerization on SH3 domain partnership. In comparison with wild type, full-length endophilin A2 heterodimers with one protomer bearing the L215D substitution exhibit very significant changes in membrane binding and shaping activities as well as a dramatic decrease of SH3 domain partnership. This suggests that subtle changes in the conformation and/or rigidity of the BAR domain impact both the control of membrane curvature and downstream binding to effectors. Finally, we show that expression, in mammalian cells, of endophilin A2 bearing the L215D substitution impairs the endocytic recycling of transferrin receptors.     

The Structure and Function of Endophilin Proteins

Members of the BAR domain protein superfamily are essential elements of cellular traffic. Endophilins are among the best studied BAR domain proteins. They have a prominent function in synaptic vesicle endocytosis (SVE), receptor trafficking and apoptosis, and in other processes that require remodeling of the membrane structure. Here, we discuss the role of endophilins in these processes and summarize novel insights into the molecular aspects of endophilin function. Also, we discuss phosphorylation of endophilins and how this and other mechanisms may contribute to disease.     

Let's go bananas: revisiting the endocytic BAR code

Against the odds of membrane resistance, members of the BIN/Amphiphysin/Rvs (BAR) domain superfamily shape membranes and their activity is indispensable for a plethora of life functions. While crystal structures of different BAR dimers advanced our understanding of membrane shaping by scaffolding and hydrophobic insertion mechanisms considerably, especially life-imaging techniques and loss-of-function studies of clathrin-mediated endocytosis with its gradually increasing curvature show that the initial idea that solely BAR domain curvatures determine their functions is oversimplified. Diagonal placing, lateral lipid-binding modes, additional lipid-binding modules, tilde shapes and formation of macromolecular lattices with different modes of organisation and arrangement increase versatility. A picture emerges, in which BAR domain proteins create macromolecular platforms, that recruit and connect different binding partners and ensure the connection and coordination of the different events during the endocytic process, such as membrane invagination, coat formation, actin nucleation, vesicle size control, fission, detachment and uncoating, in time and space, and may thereby offer mechanistic explanations for how coordination, directionality and effectiveness of a complex process with several steps and key players can be achieved.     

The crystal structure of the BAR domain from human Bin1/amphiphysin II and its implications for molecular recognition

BAR domains are found in proteins that bind and remodel membranes and participate in cytoskeletal and nuclear processes. Here, we report the crystal structure of the BAR domain from the human Bin1 protein at 2.0 A resolution. Both the quaternary and tertiary architectures of the homodimeric Bin1BAR domain are built upon "knobs-into-holes" packing of side chains, like those found in conventional left-handed coiled-coils, and this packing governs the curvature of a putative membrane-engaging concave face. Our calculations indicate that the Bin1BAR domain contains two potential sites for protein-protein interactions on the convex face of the dimer. Comparative analysis of structural features reveals that at least three architectural subtypes of the BAR domain are encoded in the human genome, represented by the Arfaptin, Bin1/Amphiphysin, and IRSp53 BAR domains. We discuss how these principal groups may differ in their potential to form regulatory heterotypic interactions.     

The Unique Mechanism of SNX9 BAR Domain for Inducing Membrane Tubulation

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins and plays a critical role in clathrin-mediated endocytosis. It has a Bin-Amphiphysin-Rvs (BAR) domain which can form a crescent-shaped homodimer structure that induces deformation of the plasma membrane. While other BAR-domain containing proteins such as amphiphysin and endophilin have an amphiphatic helix in front of the BAR domain which plays a critical role in membrane penetration, SNX9 does not. Thus, whether and how SNX9 BAR domain could induce the deformation of the plasma membrane is not clear. The present study identified the internal putative amphiphatic stretch in the 1^st α-helix of the SNX9 BAR domain and proved that together with the N-terminal helix (H₀) region, this internal putative amphiphatic stretch is critical for inducing membrane tubulation. Therefore, our study shows that SNX9 uses a unique mechanism to induce the tubulation of the plasma membrane which mediates proper membrane deformation during clathrin-mediated endocytosis.     

BAR domain proteins—a linkage between cellular membranes,signaling pathways,and the actin cytoskeleton

Actin filament assembly typically occurs in association with cellular membranes. A large number of proteins sit at the interface between actin networks and membranes, playing diverse roles such as initiation of actin polymerization, modulation of membrane curvature, and signaling. Bin/Amphiphysin/Rvs (BAR) domain proteins have been implicated in all of these functions. The BAR domain family of proteins comprises a diverse group of multi-functional effectors, characterized by their modular architecture. In addition to the membrane-curvature sensing/inducing BAR domain module, which also mediates antiparallel dimerization, most contain auxiliary domains implicated in protein-protein and/or protein-membrane interactions, including SH3, PX, PH, RhoGEF, and RhoGAP domains. The shape of the BAR domain itself varies, resulting in three major subfamilies: the classical crescent-shaped BAR, the more extended and less curved F-BAR, and the inverse curvature I-BAR subfamilies. Most members of this family have been implicated in cellular functions that require dynamic remodeling of the actin cytoskeleton, such as endocytosis, organelle trafficking, cell motility, and T-tubule biogenesis in muscle cells. Here, we review the structure and function of mammalian BAR domain proteins and the many ways in which they are interconnected with the actin cytoskeleton.     

Structural characteristics of BAR domain superfamily to sculpt the membrane

Membrane dynamics is an essential process for cell locomotion, cytokinesis, vesicular transport and organelle morphogenesis. Formation of tubes or buds from a nearly flat membrane is a widely spread feature of the membrane shape changes. The BAR (Bin/Amphiphysin/Rvs-homology) domain dimers exhibit long extended curved shapes and appear to be outstandingly suitable modules for this purpose. So far, crystal structures of 18 independent BAR domains including inverse-BAR domains and FCH-BAR domains have been reported. These atomic models show a vast variation of dimer shapes on a common and simple basic framework. Comparison of the structural variations with respect to differences in membrane sculpting properties provide useful clues about the mechanisms for modifying the framework into a particular dimer that generates and/or senses a distinct curvature set of the membrane.