A theory of ordering of elongated and curved proteins on membranes driven by density and curvature

Cell membranes interact with a myriad of curvature-active proteins that control membrane morphology and are responsible for mechanosensation and mechanotransduction. Some of these proteins, such as those containing BAR domains,...

Thermodynamics and free energy landscape of BAR-domain dimerization from molecular simulations

Nearly all proteins interact specifically with other proteins, often forming reversible bound structures whose stability is critical to function. Proteins with BAR domains function to bind to, bend, and remodel biological membranes, where the dimerization of BAR domains is a key step in this function. Here we characterize the binding thermodynamics of homodimerization between the LSP1 BAR domain proteins in solution, using Molecular Dynamics (MD) simulations. By combining the MARTINI coarse-grained protein models with enhanced sampling through metadynamics, we construct a two-dimensional free energy surface quantifying the bound versus unbound ensembles as a function of two distance variables. Our simulations portray a heterogeneous and extraordinarily stable bound ensemble for these modeled LSP1 proteins. The proper crystal structure dimer has a large hydrophobic interface that is part of a stable minima on the free energy surface, which is enthalpically the minima of all bound structures. However, we also find several other stable nonspecific dimers with comparable free energies to the specific dimer. Through structure-based clustering of these bound structures, we find that some of these ‘nonspecific’ contacts involve extended tail regions that help stabilize the higher-order oligomers formed by BAR-domains, contacts that are separated from the homodimer interface. We find that the known membrane-binding residues of the LSP1 proteins rarely participate in any of the bound interfaces, but that both patches of residues are aligned to interact with the membrane in the specific dimer. Hence, we would expect a strong selection of the specific dimer in binding to the membrane. The effect of a 100mM NaCl buffer reduces the relative stability of nonspecific dimers compared to the specific dimer, indicating that it would help prevent aggregation of the proteins. With these results, we provide the first free energy characterization of interaction pathways in this important class of membrane sculpting domains, revealing a variety of interfacial contacts outside of the specific dimer that may help stabilize its oligomeric assemblies.

Recycling of autophagosomal components from autolysosomes by recycler complex

Autolysosomes contain components both from autophagosomes and lysosomes. The contents inside the autophagosomal lumen are degraded during autophagy, while the fate of autophagosomal components on autolysosomal membrane remains unknown. Here, we found the autophagosomal membrane and transmembrane proteins are not degraded, but recycled from autolysosomes. We named this process autophagosomal components recycling (ACR). We further identified a multiprotein complex composed of SNX4, SNX5 and SNX17 essential for ACR which we termed “recycler”. In this, SNX4 and SNX5 form a heterodimer that recognizes an autophagosomal cargo STX17 and is required for generating membrane curvature on autolysosomes both via their BAR domains, to mediate the cargo sorting process. SNX17 interacts with both the dynein-dynactin complex and the SNX4-SNX5 dimer to facilitate retrograde transport of STX17. Depletion of any subunit of recycler completely blocks ACR, and also inhibits autophagy. Our discovery of ACR and identification of recycler reveal an important retrieval and recycling pathway on autolysosomes.

Mechanism of negative membrane curvature generation by I-BAR domains

The membrane sculpting ability of BAR domains has been attributed to the intrinsic curvature of their banana-shaped dimeric structure. However, there is often a mismatch between this intrinsic curvature and the diameter of the membrane tubules generated. I-BAR domains have been especially mysterious: they are almost flat but generate high negative membrane curvature. Here, we use atomistic implicit-solvent computer modeling to show that the membrane bending of the IRSP53 I-BAR domain is dictated by its higher oligomeric structure, whose curvature is completely unrelated to the intrinsic curvature of the dimer. Two other I-BARs gave similar results, whereas a flat F-BAR sheet developed a concave membrane binding interface, consistent with its observed positive membrane curvature generation. Laterally interacting helical spirals of I-BAR dimers on tube interiors are stable and have an enhanced binding energy that is sufficient for membrane bending to experimentally observed tubule diameters at a reasonable surface density.

Membrane-mediated forces can stabilize tubular assemblies of I-Bar proteins

Collective action by Inverse-BAR (I-BAR) domains drive micron-scale membrane remodeling. The macroscopic curvature sensing and generation behavior of I-BAR domains is well characterized, and computational models have suggested various mechanisms on simplified membrane systems, but there remain missing connections between the complex environment of the cell and the models proposed thus far. Here, we show a connection between the role of protein curvature and lipid clustering in the stabilization of large membrane deformations. We find lipid clustering provides a directional membrane-mediated interaction between membrane-bound I-BAR domains. Lipid clusters stabilize I-BAR domain aggregates that would not arise through membrane fluctuation-based or curvature-based interactions. Inside of membrane protrusions, lipid cluster-mediated interaction draws long side-by-side aggregates together resulting in more cylindrical protrusions as opposed to bulbous, irregularly shaped protrusions.Statement of SignificanceMembrane remodeling occurs throughout the cell and is crucial to proper cellular function. In the cellular environment, I-BAR proteins are responsible for sensing membrane curvature and initiating the formation of protrusions outward from the cell. Additionally, there is a large body of evidence that I-BAR domains are sufficient to reshape the membrane on scales much larger than any single domain. The mechanism by which I-BAR domains can remodel the membrane is uncertain. However, experiments show that membrane composition and most notably negatively-charge lipids like PIP2 play a role in the onset of tubulation. Using coarse-grained models, we show that I-BAR domains can cluster negatively charge lipids and clustered PIP2-like membrane structures facilitate a directional membrane-mediated interaction between I-BAR domains.

Quantitative investigation of negative membrane curvature sensing and generation by I-BARs in filopodia of living cells

We analyze diffraction-limited filopodia of living cells to quantify negative curvature sensing and generation for two prototypic I-BAR domains.

BAR scaffolds drive membrane fission by crowding disordered domains

Cellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysin-rvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of isolated BAR domains in vitro, the current paradigm is that BAR domain–containing proteins polymerize into cylindrical scaffolds that stabilize lipid tubules. But in nature, proteins that contain BAR domains often also contain large intrinsically disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain–containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data suggest that the ability to concentrate disordered domains is a key driver of membrane remodeling and fission by BAR domain–containing proteins.

BAR scaffolds drive membrane fission by crowding disordered domains

SummaryCylindrical protein scaffolds are thought to stabilize membrane tubules, preventing membrane fission. In contrast, Snead et al. find that when scaffold proteins assemble, bulky disordered domains within them become acutely concentrated, generating steric pressure that destabilizes tubules, driving fission.AbstractCellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysinrvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of BAR domains in isolation, the current paradigm is that they polymerize into cylindrical scaffolds that stabilize lipid tubules, preventing membrane fission. But in nature BAR domains are often part of multi-domain proteins that contain large intrinsically-disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data challenge the idea that cellular membranes adopt the curvature of BAR scaffolds, suggesting instead that the ability to concentrate disordered domains is the key requirement for membrane remodeling and fission by BAR domain-containing proteins.