A Hinge in the Distal End of the PACSIN 2 F-BAR Domain May Contribute to Membrane-Curvature Sensing

The protein kinase C and casein kinase 2 substrates in neurons (PACSINs) represent a subfamily of membrane-binding proteins characterized by an amino-terminal Bin-Amphiphysin-Rvs (F-BAR) domain. PACSINs link membrane trafficking with actin dynamics and regulate the localization of distinct cargo molecules. The F-BAR domain forms a dimer essential for lipid binding. We have obtained crystals of authentic murine PACSIN 2 that contain an ordered F-BAR domain, indicating that additional domains are flexibly connected to F-BAR. The structure shares similarity to other BAR domains and exhibits special features unique to PACSINs. These include the uneven distribution of charged residues on the concave molecular surface and a so-called wedge loop that is driven into the membrane upon binding of PACSIN. The murine PACSIN 2 F-BAR domain requires dimerization for sensing of curved membranes, and the present structure also provides a mechanism for higher-order oligomer formation. Importantly, comparison of murine with human and Drosophila PACSIN 2 F-BAR domains reveals stark differences in the orientation of distal helical segments leading to a wider crescent shape of murine PACSIN 2. We define hinge residues for these movements that may help PACSINs sense and concomitantly reinforce membrane curvature.     

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.     

A Highlights from MBoC Selection: Formation of membrane ridges and scallops by the F-BAR protein Nervous Wreck

Eukaryotic cells are defined by extensive intracellular compartmentalization, which requires dynamic membrane remodeling. FER/Cip4 homology-Bin/amphiphysin/Rvs (F-BAR) domain family proteins form crescent-shaped dimers, which can bend membranes into buds and tubules of defined geometry and lipid composition. However, these proteins exhibit an unexplained wide diversity of membrane-deforming activities in vitro and functions in vivo. We find that the F-BAR domain of the neuronal protein Nervous Wreck (Nwk) has a novel higher-order structure and membrane-deforming activity that distinguishes it from previously described F-BAR proteins. The Nwk F-BAR domain assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. This activity depends on structural determinants at the tips of the F-BAR dimer and on electrostatic interactions of the membrane with the F-BAR concave surface. In cells, Nwk-induced scallops can be extended by cytoskeletal forces to produce protrusions at the plasma membrane. Our results define a new F-BAR membrane-deforming activity and illustrate a molecular mechanism by which positively curved F-BAR domains can produce a variety of membrane curvatures. These findings expand the repertoire of F-BAR domain mediated membrane deformation and suggest that unique modes of higher-order assembly can define how these proteins sculpt the membrane.     

Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins

Cell membranes undergo continuous curvature changes as a result of membrane trafficking and cell motility. Deformations are achieved both by forces extrinsic to the membrane as well as by structural modifications in the bilayer or at the bilayer surface that favor the acquisition of curvature. We report here that a family of proteins previously implicated in the regulation of the actin cytoskeleton also have powerful lipid bilayer-deforming properties via an N-terminal module (F-BAR) similar to the BAR domain. Several such proteins, like a subset of BAR domain proteins, bind to dynamin, a GTPase implicated in endocytosis and actin dynamics, via SH3 domains. The ability of BAR and F-BAR domain proteins to induce tubular invaginations of the plasma membrane is enhanced by disruption of the actin cytoskeleton and is antagonized by dynamin. These results suggest a close interplay between the mechanisms that control actin dynamics and those that mediate plasma membrane invagination and fission.     

Subcellular membrane curvature mediated by the BAR domain superfamily proteins

The Bin-Amphiphysin-Rvs167 (BAR) domain superfamily consists of proteins containing the BAR domain, the extended FCH (EFC)/FCH-BAR (F-BAR) domain, or the IRSp53-MIM homology domain (IMD)/inverse BAR (I-BAR) domain. These domains bind membranes through electrostatic interactions between the negative charges of the membranes and the positive charges on the structural surface of homo-dimeric BAR domain superfamily members. Some BAR superfamily members have membrane-penetrating insertion loops, which also contribute to the membrane binding by the proteins. The membrane-binding surface of each BAR domain superfamily member has its own unique curvature that governs or senses the curvature of the membrane for BAR-domain binding. The wide range of BAR-domain surface curvatures correlates with the various invaginations and protrusions of cells. Therefore, each BAR domain superfamily member may generate and recognize the curvature of the membrane of each subcellular structure, such as clathrin-coated pits or filopodia. The BAR domain superfamily proteins may regulate their own catalytic activity or that of their binding proteins, depending on the membrane curvature of their corresponding subcellular structures.     

The eisosome core is composed of BAR domain proteins

Eisosomes define sites of plasma membrane organization. In Saccharomyces cerevisiae, eisosomes delimit furrow-like plasma membrane invaginations that concentrate sterols, transporters, and signaling molecules. Eisosomes are static macromolecular assemblies composed of cytoplasmic proteins, most of which have no known function. In this study, we used a bioinformatics approach to analyze a set of 20 eisosome proteins. We found that the core components of eisosomes, paralogue proteins Pil1 and Lsp1, are distant homologues of membrane-sculpting Bin/amphiphysin/Rvs (BAR) proteins. Consistent with this finding, purified recombinant Pil1 and Lsp1 tubulated liposomes and formed tubules when the proteins were overexpressed in mammalian cells. Structural homology modeling and site-directed mutagenesis indicate that Pil1 positively charged surface patches are needed for membrane binding and liposome tubulation. Pil1 BAR domain mutants were defective in both eisosome assembly and plasma membrane domain organization. In addition, we found that eisosome-associated proteins Slm1 and Slm2 have F-BAR domains and that these domains are needed for targeting to furrow-like plasma membrane invaginations. Our results support a model in which BAR domain protein-mediated membrane bending leads to clustering of lipids and proteins within the plasma membrane.     

Role of helix 0 of the N-BAR domain in membrane curvature generation

A group of proteins with cell membrane remodeling properties is also able to change dramatically the morphology of liposomes in vitro, frequently inducing tubulation. For a number of these proteins, the mechanism by which this effect is exerted has been proposed to be the embedding of amphipathic helices into the lipid bilayer. For proteins presenting BAR domains, removal of an N-terminal amphipathic α-helix (H0-NBAR) results in much lower membrane tubulation efficiency, pointing to a fundamental role of this protein segment. Here, we studied the interaction of a peptide corresponding to H0-NBAR with model lipid membranes. H0-NBAR bound avidly to anionic liposomes but partitioned weakly to zwitterionic bilayers, suggesting an essentially electrostatic interaction with the lipid bilayer. Interestingly, it is shown that after membrane incorporation, the peptide oligomerizes as an antiparallel dimer, suggesting a potential role of H0-NBAR in the mediation of BAR domain oligomerization. Through monitoring the effect of H0-NBAR on liposome shape by cryoelectron microscopy, it is clear that membrane morphology is not radically changed. We conclude that H0-NBAR alone is not able to induce vesicle curvature, and its function must be related to the promotion of the scaffold effect provided by the concave surface of the BAR domain.     

Four-scale description of membrane sculpting by BAR domains

BAR domains are proteins that sense and sculpt curved membranes in cells, furnishing a relatively well-studied example of mechanisms employed in cellular morphogenesis. We report a computational study of membrane bending by BAR domains at four levels of resolution, described by 1), all-atom molecular dynamics; 2), residue-based coarse-graining (resolving single amino acids and lipid molecules); 3), shape-based coarse-graining (resolving overall protein and membrane shapes); and 4), a continuum elastic membrane model. Membrane sculpting performed by BAR domains collectively is observed in agreement with experiments. Different arrangements of BAR domains on the membrane surface are found to lead to distinct membrane curvatures and bending dynamics.     

Structural basis for membrane binding specificity of the Bin/Amphiphysin/Rvs (BAR) domain of Arfaptin-2 determined by Arl1 GTPase

Membrane-sculpting BAR (Bin/Amphiphysin/Rvs) domains form a crescent-shaped homodimer that can sense and induce membrane curvature through its positively charged concave face. We have recently shown that Arfaptin-2, which was originally identified as a binding partner for the Arf and Rac1 GTPases, binds to Arl1 through its BAR domain and is recruited onto Golgi membranes. There, Arfaptin-2 induces membrane tubules. Here, we report the crystal structure of the Arfaptin-2 BAR homodimer in complex with two Arl1 molecules bound symmetrically to each side, leaving the concave face open for membrane association. The overall structure of the Arl1·Arfaptin-2 BAR complex closely resembles that of the PX-BAR domain of sorting nexin 9, suggesting similar mechanisms underlying BAR domain targeting to specific organellar membranes. The Arl1·Arfaptin-2 BAR structure suggests that one of the two Arl1 molecules competes with Rac1, which binds to the concave face of the Arfaptin-2 BAR homodimer and may hinder its membrane association.     

Rigidity of wedge loop in PACSIN 3 protein is a key factor in dictating diameters of tubules

BAR (Bin/amphiphysin/Rvs) domain-containing proteins participate in cellular membrane remodeling. The F-BAR proteins normally generate low curvature tubules. However, in the PACSIN subfamily, the F-BAR domain from PACSIN 1 and 2 can induce both high and low curvature tubules. We found that unlike PACSIN 1 and 2, PACSIN 3 could only induce low curvature tubules. To elucidate the key factors that dictate the tubule curvature, crystal structures of all three PACSIN F-BAR domains were determined. A novel type of lateral interaction mediated by a wedge loop is observed between the F-BAR neighboring dimers. Comparisons of the structures of PACSIN 3 with PACSIN 1 and 2 indicate that the wedge loop of PACSIN 3 is more rigid, which influences the lateral interactions between assembled dimers. We further identified the residues that affect the rigidity of the loop by mutagenesis and determined the structures of two PACSIN 3 wedge loop mutants. Our results suggest that the rigidity-mediated conformations of the wedge loop correlate well with the various crystal packing modes and membrane tubulations. Thus, the rigidity of the wedge loop is a key factor in dictating tubule diameters.     

Factors influencing local membrane curvature induction by N-BAR domains as revealed by molecular dynamics simulations

N-BAR domains are protein modules that bind to and induce curvature in membranes via a charged concave surface and N-terminal amphipathic helices. Recently, molecular dynamics simulations have demonstrated that the N-BAR domain can induce a strong local curvature that matches the curvature of the BAR domain surface facing the bilayer. Here we present further molecular dynamics simulations that examine in greater detail the roles of the concave surface and amphipathic helices in driving local membrane curvature. We find that the strong curvature induction observed in our previous simulations requires the stable presentation of the charged concave surface to the membrane and is not driven by the membrane-embedded amphipathic helices. Nevertheless, without these amphipathic helices embedded in the membrane, the N-BAR domain does not maintain a close association with the bilayer, and fails to drive membrane curvature. Increasing the membrane negative charge through the addition of PIP₂ facilitates closer association with the membrane in the absence of embedded helices. At sufficiently high concentrations, amphipathic helices embedded in the membrane drive membrane curvature independently of the BAR domain.     

Flexible scaffolding made of rigid BARs

Crescent-shaped BAR domains are generic actors in the creation of membrane curvature. In this issue, Frost et al. (2008) reveal how collective twisting of rigid F-BAR domains on a soft membrane surface may lead to different membrane curvatures.     

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.     

Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature

A spectrum of membrane curvatures exists within cells, and proteins have evolved different modules to detect, create, and maintain these curvatures. Here we present the crystal structure of one such module found within human FCHo2. This F-BAR (extended FCH) module consists of two F-BAR domains, forming an intrinsically curved all-helical antiparallel dimer with a Kd of 2.5 microM. The module binds liposomes via a concave face, deforming them into tubules with variable diameters of up to 130 nm. Pulse EPR studies showed the membrane-bound dimer is the same as the crystal dimer, although the N-terminal helix changed conformation on membrane binding. Mutation of a phenylalanine on this helix partially attenuated narrow tubule formation, and resulted in a gain of curvature sensitivity. This structure shows a distant relationship to curvature-sensing BAR modules, and suggests how similar coiled-coil architectures in the BAR superfamily have evolved to expand the repertoire of membrane-sculpting possibilities.     

BAR, F-BAR (EFC) and ENTH/ANTH domains in the regulation of membrane-cytosol interfaces and membrane curvature

BAR and ENTH domains are families of alpha-helical lipid bilayer binding modules found in proteins that function in endocytosis, actin regulation and signaling. Several members of these families not only bind the bilayer, but also participate in the regulation of its curvature. These properties are thought to play physiological roles at sites of membrane budding and at other sites where narrow tubular membranes occur in vivo. Studies of BAR and ENTH domains and of their flanking regions have provided new insights into mechanisms of membrane deformation and curvature sensing, and have emphasized the importance of amphipathic helices, thought to intercalate in one of the leaflets of the lipid bilayer, in the generation of membrane curvature. Structural studies and database searches are rapidly expanding the BAR and ENTH domains families, with the identification of new related domains and subfamilies, such as F-BAR (also called EFC) domains and ANTH domains, respectively. Here we present a short overview of the properties of these domains based on evidence obtained from genetics, cell biology, biochemistry and structural biology.     

Membrane curvature: how BAR domains bend bilayers

An important new structure suggests the BAR domain is a membrane-binding module that can both produce and sense membrane curvature. BAR resembles a banana that binds membranes electrostatically through its positively charged, concave surface.     

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.     

Exploring the binding dynamics of BAR proteins

We used a continuum model based on the Helfrich free energy to investigate the binding dynamics of a lipid bilayer to a BAR domain surface of a crescent-like shape of positive (e.g. I-BAR shape) or negative (e.g. F-BAR shape) intrinsic curvature. According to structural data, it has been suggested that negatively charged membrane lipids are bound to positively charged amino acids at the binding interface of BAR proteins, contributing a negative binding energy to the system free energy. In addition, the cone-like shape of negatively charged lipids on the inner side of a cell membrane might contribute a positive intrinsic curvature, facilitating the initial bending towards the crescent-like shape of the BAR domain. In the present study, we hypothesize that in the limit of a rigid BAR domain shape, the negative binding energy and the coupling between the intrinsic curvature of negatively charged lipids and the membrane curvature drive the bending of the membrane. To estimate the binding energy, the electric potential at the charged surface of a BAR domain was calculated using the Langevin-Bikerman equation. Results of numerical simulations reveal that the binding energy is important for the initial instability (i.e. bending of a membrane), while the coupling between the intrinsic shapes of lipids and membrane curvature could be crucial for the curvature-dependent aggregation of negatively charged lipids near the surface of the BAR domain. In the discussion, we suggest novel experiments using patch clamp techniques to analyze the binding dynamics of BAR proteins, as well as the possible role of BAR proteins in the fusion pore stability of exovesicles.     

Biochemical and functional significance of F-BAR domain proteins interaction with WASP/N-WASP

The Bin-Amphiphysin-Rvs (BAR) domain family of proteins includes groups which promote positive (classical BAR, N-BAR, and F-BAR) and negative (I-BAR) membrane deformation. Of these groups, the F-BAR subfamily is the most diverse in its biochemical properties. F-BAR domain proteins dimerize to form a tight scaffold about the membrane. The F-BAR domain provides a banana-shaped, alpha-helical structure that senses membrane curvature. Different types of F-BAR domain proteins contain tyrosine kinase or GTPase activities; some interact with phosphatases and RhoGTPases. Most possess an SH3 domain that facilitates the recruitment and activation of WASP/N-WASP. Thus, F-BAR domain proteins affect remodeling of both membrane and the actin cytoskeleton. The purpose of this review is to highlight the role of F-BAR proteins in coupling WASP/N-WASP to cytoskeletal remodeling. A role for F-BAR/WASP interaction in human diseases affecting nervous, blood, and neoplastic tissues is discussed.     

Modeling Membrane Deformations and Lipid Demixing upon Protein-Membrane Interaction: The BAR Dimer Adsorption

We use a self-consistent mean-field theory, designed to investigate membrane reshaping and lipid demixing upon interaction with proteins, to explore BAR domains interacting with large patches of lipid membranes of heterogeneous compositions. The computational model includes contributions to the system free energy from electrostatic interactions and elastic energies of the membrane, as well as salt and lipid mixing entropies. The results from our simulation of a single adsorbing Amphiphysin BAR dimer indicate that it is capable of stabilizing a significantly curved membrane. However, we predict that such deformations will occur only for membrane patches that have the inherent propensity for high curvature, reflected in the tendency to create local distortions that closely match the curvature of the BAR dimer itself. Such favorable preconditioning for BAR-membrane interaction may be the result of perturbations such as local lipid demixing induced by the interaction, or of a prior insertion of the BAR domain's amphiphatic N-helix. From our simulations it appears that local segregation of charged lipids under the influence of the BAR dimer cannot produce high enough asymmetry between bilayer leaflets to induce significant bending. In the absence of additional energy contributions that favor membrane asymmetry, the membrane will remain nearly flat upon single BAR dimer adsorption, relative to the undulation expected from thermal fluctuations. Thus, we conclude that the N-helix insertions have a critical mechanistic role in the local perturbation and curving of the membrane, which is then stabilized by the electrostatic interaction with the BAR dimer. We discuss how these results can be used to estimate the tendency of BARs to bend membranes in terms of a spatially nonisotropic spontaneous curvature.