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.

Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the bin/amphiphysin/rvs domain (BAR) proteins. Many open questions remain about the mechanisms by which membrane-bending proteins function. Addressing this shortfall, recent structures of BAR protein:membrane complexes support existing mechanistic models, but also produced novel insights into how BAR-domain proteins sense, stabilize and generate curvature. Here we review these recent findings, focusing on how BAR proteins interact with the membrane, and how the resulting scaffold structures might aid the recruitment of other proteins to the sites where membranes are bent.

The ubiquitin-proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and mis-folded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures bring some surprising insights into the functional mechanism of the proteasome and will provide invaluable guidance for genetic and biochemical studies of this key regulatory system.

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1’s own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.

Mesoscopic simulations and electron microscopy of N-BAR domain-induced liposome remodeling are used to characterize the process of liposome tubulation and vesiculation. The overall process of membrane remodeling is found to involve complex couplings among the N-BAR protein density, the degree of N-BAR oligomerization, and the membrane density. A comparison of complex remodeled liposome structures from mesoscopic simulations with those measured by electron microscopy experiments suggests that the process of membrane remodeling can be described via an appropriate mesoscopic free energy framework. Liposome remodeling more representative of F-BAR domains is also presented within the mesoscopic simulation framework.

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-μs time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 μM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na+-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at −90 mV). Applying step changes to the transmembrane potential, V m, of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a τ of ∼15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<−40 mV) and activation at positive V m (>0 mV). A similar inhibitory effect at V m < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V m curve. Jumping the glutamate concentration to 100 μM generated biphasic, saturable transient transport and anion currents (K m ∼ 5 μM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1–3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.

Endophilin N-BAR (N-terminal helix and Bin/amphiphysin/Rvs) domain tubulates and vesiculates lipid membranes in vitro via its crescent-shaped dimer and four amphipathic helices that penetrate into membranes as wedges. Like F-BAR domains, endophilin N-BAR also forms a scaffold on membrane tubes. Unlike F-BARs, endophilin N-BARs have N-terminal H0 amphipathic helices that are proposed to interact with other N-BARs in oligomer lattices. Recent cryo-electron microscopy reconstructions shed light on the organization of the N-BAR lattice coats on a nanometer scale. However, because of the resolution of the reconstructions, the precise positioning of the amphipathic helices is still ambiguous. In this work, we applied a coarse-grained model to study various membrane remodeling scenarios induced by endophilin N-BARs. We found that H0 helices of N-BARs prefer to align in an antiparallel manner at two ends of the protein to form a stable lattice. The deletion of H0 helices causes disruption of the lattice. In addition, we analyzed the persistence lengths of the protein-coated tubes and found that the stiffness of endophilin N-BAR-coated tubules qualitatively agrees with previous experimental work studying N-BAR-coated tubules. Large-scale simulations on membrane liposomes revealed a systematic relation between H0 helix density and local membrane curvature fluctuations. The data also suggest that the H0 helix is required for BARs to form organized structures on the liposome, further illustrating its important function.

Key cellular processes are frequently accompanied by protein-facilitated shape changes in the plasma membrane. N-BAR-domain protein modules generate curvature by means of complex interactions with the membrane surface. The way they assemble and the mechanism by which they operate are largely dependent on their binding density. Although the mechanism at lower densities has recently begun to emerge, how membrane scaffolds form at high densities remains unclear. By combining electron microscopy and multiscale simulations, we show that N-BAR proteins at high densities can transform a lipid vesicle into a 3D tubular network. We show that this process is a consequence of excess adhesive energy combined with the local stiffening of the membrane, which occurs in a narrow range of mechanical properties of both the membrane and the protein. We show that lipid diffusion is significantly reduced by protein binding at this density regime and even more in areas of high Gaussian curvature, indicating a potential effect on molecular transport in cells. Finally, we reveal that the breaking of the bilayer topology is accompanied by the nematic arrangement of the protein on the surface, a structural motif that likely drives the formation of reticular structures in living cells.