Biomimetic Vesicles with Designer Phospholipids Can Sense Environmental Redox Cues.

Cell-like materials that sense environmental cues can serve as next-generation biosensors and help advance the understanding of intercellular communication. Currently, bottom-up engineering of protocell models from molecular building blocks remains a grand challenge chemists face. Herein, we describe giant unilamellar vesicles (GUVs) with biomimetic lipid membranes capable of sensing environmental redox cues. The GUVs employ activity-based sensing through designer phospholipids that are fluorescently activated in response to specific reductive (hydrogen sulfide) or oxidative (hydrogen peroxide) conditions. These synthetic phospholipids are derived from 1,2-dipalmitoyl-rac-glycero-3-phosphocholine and they possess a headgroup with heterocyclic aromatic motifs. Despite their structural deviation from the phosphocholine headgroup, the designer phospholipids (0.5-1.0 mol %) mixed with natural lipids can vesiculate, and the resulting GUVs (7-20 µm in diameter) remain intact over the course of redox sensing. All-atom molecular dynamics simulations gave insight into how these lipids are positioned within the hydrophobic core of the membrane bilayer and at the membrane-water interface. This work provides a purely chemical method to investigate potential redox signaling and opens up new design opportunities for soft materials that mimic protocells.

Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

Magnesium Transporter MgtA revealed as a Dimeric P-type ATPase.

Magnesium (Mg2+) uptake systems are present in all domains of life given the vital role of this ion. Bacteria acquire Mg2+ via conserved Mg2+ channels and transporters. The transporters are required for growth when Mg2+ is limiting or during bacterial pathogenesis, but, despite their significance, there are no known structures for these transporters. Here we report the first structure of the Mg2+ transporter MgtA solved by single particle cryo-electron microscopy (cryo-EM). Using mild membrane extraction, we obtained high resolution structures of both a homodimeric form (2.9 Å), the first for a P-type ATPase, and a monomeric form (3.6 Å). Each monomer unit of MgtA displays a structural architecture that is similar to other P-type ATPases with a transmembrane domain and two soluble domains. The dimer interface consists of contacts between residues in adjacent soluble nucleotide binding and phosphotransfer regions of the haloacid dehalogenase (HAD) domain. We suggest oligomerization is a conserved structural feature of the diverse family of P-type ATPase transporters. The ATP binding site and conformational dynamics upon nucleotide binding to MgtA were characterized using a combination of cryo-EM, molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and mutagenesis. Our structure also revealed a Mg2+ ion in the transmembrane segments, which, when combined with sequence conservation and mutagenesis studies, allowed us to propose a model for Mg2+ transport across the lipid bilayer. Finally, our work revealed the N-terminal domain structure and cytoplasmic Mg2+ binding sites, which have implications for related P-type ATPases defective in human disease.

Synaptotagmin 7 C2 domains induce membrane curvature stress via electrostatic interactions and the wedge mechanism.

Synaptotagmin 7 (Syt-7) is part of the synaptotagmin protein family that regulates exocytotic lipid membrane fusion. Among the family, Syt-7 stands out by its membrane binding strength and stabilization of long-lived membrane fusion pores. Given that Syt-7 vesicles form long-lived fusion pores, we hypothesize that its interactions with the membrane stabilize the specific curvatures, thicknesses, and lipid compositions that support a metastable fusion pore. Using all-atom molecular dynamics simulations and FRET-based assays of Syt-7's membrane-binding C2 domains (C2A and C2B), we found that Syt-7 C2 domains sequester anionic lipids, are sensitive to cholesterol, thin membranes, and generate lipid membrane curvature by two competing, but related mechanisms. First, Syt-7 forms strong electrostatic contacts with the membrane, generating negative curvature stress. Second, Syt-7's calcium binding loops embed in the membrane surface, acting as a wedge to thin the membrane and induce positive curvature stress. These curvature mechanisms are linked by the protein insertion depth as well as the resulting protein tilt. Simplified quantitative models of the curvature-generating mechanisms link simulation observables to their membrane-reshaping effectiveness.

Simulated dynamic cholesterol redistribution favors membrane fusion pore constriction.

Endo- and exocytosis proceed through a highly strained membrane fusion pore topology regardless of the aiding protein machinery. The membrane's lipid components bias fusion pores toward expansion or closure, modifying the necessary work done by proteins. Cholesterol, a key component of plasma membranes, promotes both inverted lipid phases with concave leaflets (i.e., negative total curvature, which thins the leaflet) and flat bilayer phases with thick, ordered hydrophobic interiors. We demonstrate by theory and simulation that both leaflets of nascent catenoidal fusion pores have negative total curvature. Furthermore, the hydrophobic core of bilayers with strong negative Gaussian curvature is thinned. Therefore, it is an open question whether cholesterol will be enriched in these regions because of the negative total curvature or depleted because of the membrane thinning. Here, we compare all-atom molecular dynamics simulations (built using a procedure to create specific fusion pore geometries) and theory to understand the underlying reasons for lipid redistribution on fusion pores. Our all-atom molecular dynamics simulations resolve this question by showing that cholesterol is strongly excluded from the thinned neck of fusion and fission pores, revealing that thickness (and/or lipid order) influences cholesterol distributions more than curvature. The results imply that cholesterol exclusion can drive fusion pore closure by creating a small, cholesterol-depleted zone in the neck. This model agrees with literature evidence that membrane reshaping is connected to cholesterol-dependent lateral phase separation.

Physiological changes in bilayer thickness induced by cholesterol control GPCR rhodopsin function.

We monitored the effect on function of the G-protein-coupled receptor (GPCR) rhodopsin from small, stepwise changes in bilayer thickness induced by cholesterol. Over a range of phosphatidylcholine bilayers with hydrophobic thickness from ≈21 Å to 38 Å, the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium was monitored with UV-visible spectroscopy while ordering of hydrocarbon chains was probed by 2H-NMR. Addition of cholesterol shifted equilibrium toward MII for bilayers thinner than the average length of hydrophobic transmembrane helices (27 Å) and to MI for thicker bilayers, while small bilayer thickness changes within the range of the protein hydrophobic thickness drastically up- or downregulated MII formation. The cholesterol-induced shifts toward MII for thinner membranes correlated with the cholesterol-induced increase of bilayer hydrophobic thickness measured by NMR, consistent with continuum elastic modeling. The energetic penalty of adding cholesterol to thick bilayers caused rhodopsin oligomerization and a shift toward MI. In membranes of physiological thickness, changes in bilayer mechanical properties induced by cholesterol potentiated the interplay between bilayer and protein thickness resulting in large swings of the MI-MII equilibrium. In membrane containing cholesterol, elastic deformations near the protein are a dominant energetic contribution to the functional equilibrium of the model GPCR rhodopsin.

Softening in two-component lipid mixtures by spontaneous curvature variance.

The bending modulus of a lipid bilayer quantifies its mechanical resistance to curvature. It is typically understood in terms of thickness, e.g., thicker bilayers are stiffer. Here, we describe an additional and powerful molecular determinant of stiffness - the variance in the distribution of curvature sensitivity of lipids and lipid conformations. Zwitterionic choline and ethanolamine head-groups of glycero-phospholipids dynamically explore inter- and intra-species interactions, leading to transient clustering. We demonstrate that these clusters couple strongly to negative curvature, exciting undulatory membrane modes and reducing the apparent bending modulus. Three forcefields (Martini 2, Martini 3, and all-atom CHARMM C36) each show the effect to a different extent, with the coarse-grained Martini models showing the most clustering and thus the most softening. The theory is a guide to understanding the stiffness of biological membranes with their complex composition, as well as how choices of forcefield parameterization are translated into mechanical stiffness.

Molecular mechanisms of spontaneous curvature and softening in complex lipid bilayer mixtures.

Membrane reshaping is an essential biological process. The chemical composition of lipid membranes determines their mechanical properties and thus the energetics of their shape. Hundreds of distinct lipid species make up native bilayers, and this diversity complicates efforts to uncover what compositional factors drive membrane stability in cells. Simplifying assumptions, therefore, are used to generate quantitative predictions of bilayer dynamics based on lipid composition. One assumption commonly used is that "per lipid" mechanical properties are both additive and constant-that they are an intrinsic property of lipids independent of the surrounding composition. Related to this is the assumption that lipid bulkiness, or "shape," determines its curvature preference, independently of context. In this study, all-atom molecular dynamics simulations on three separate multilipid systems were used to explicitly test these assumptions, applying methodology recently developed to isolate properties of single lipids or nanometer-scale patches of lipids. The curvature preference experienced by populations of lipid conformations were inferred from their redistribution on a dynamically fluctuating bilayer. Representative populations were extracted by both structural similarity and semi-automated hidden Markov model analysis. The curvature preferences of lipid dimers were then determined and compared with an additive model that combines the monomer curvature preference of both the individual lipids. In all three systems, we identified conformational subpopulations of lipid dimers that showed non-additive curvature preference, in each case mediated by a special chemical interaction (e.g., hydrogen bonding). Our study highlights the importance of specific chemical interactions between lipids in multicomponent bilayers and the impact of interactions on bilayer stiffness. We identify two mechanisms of bilayer softening: diffusional softening, driven by the dynamic coupling between lipid distributions and membrane undulations, and conformational softening, driven by the inter-conversion between distinct dimeric conformations.

Cholesterol Binds the Amphipathic Helix of IFITM3 and Regulates Antiviral Activity.

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.

Large self-assembled clathrin lattices spontaneously disassemble without sufficient adaptor proteins.

Clathrin-coated structures must assemble on cell membranes to internalize receptors, with the clathrin protein only linked to the membrane via adaptor proteins. These structures can grow surprisingly large, containing over 20 clathrin, yet they often fail to form productive vesicles, instead aborting and disassembling. We show that clathrin structures of this size can both form and disassemble spontaneously when adaptor protein availability is low, despite high abundance of clathrin. Here, we combine recent in vitro kinetic measurements with microscopic reaction-diffusion simulations and theory to differentiate mechanisms of stable vs unstable clathrin assembly on membranes. While in vitro conditions drive assembly of robust, stable lattices, we show that concentrations, geometry, and dimensional reduction in physiologic-like conditions do not support nucleation if only the key adaptor AP-2 is included, due to its insufficient abundance. Nucleation requires a stoichiometry of adaptor to clathrin that exceeds 1:1, meaning additional adaptor types are necessary to form lattices successfully and efficiently. We show that the critical nucleus contains ~25 clathrin, remarkably similar to sizes of the transient and abortive structures observed in vivo. Lastly, we quantify the cost of bending the membrane under our curved clathrin lattices using a continuum membrane model. We find that the cost of bending the membrane could be largely offset by the energetic benefit of forming curved rather than flat structures, with numbers comparable to experiments. Our model predicts how adaptor density can tune clathrin-coated structures from the transient to the stable, showing that active energy consumption is therefore not required for lattice disassembly or remodeling during growth, which is a critical advance towards predicting productive vesicle formation.

Observed steric crowding at modest coverage requires a particular membrane-binding scheme or a complementary mechanism.

Membrane shape transitions, including fusion and fission, play an important role in many biological processes. It is therefore essential to understand mechanisms of "curvature generation," the mathematical quantification of membrane shape. Among the different mechanisms is the effect of steric pressure between proteins crowded on a surface. At a higher curvature, there is more space for the crowders and less steric pressure. Currently, the physical model of curvature induction by crowding views the proteins as being bound to the surface as a whole rather than to the underlying lipids. Here, we split the previously understood model into two pieces: first, the reduction in steric pressure due to reduced collisions between proteins, and second, the increased area available to the protein that is independent of other crowders. The cases are distinguished by how the crowder is attached to the membrane. When a protein is attached to a specific lipid, as is the case in a typical crowding experiment, one should not model its lateral entropy; this has already been accounted for by the underlying lipid. The Carnahan-Starling pressure includes this lateral entropy. The revised theory predicts that a purely entropic crowding mechanism is inconsistent with observations of reshaping at the lower range of surface coverage, suggesting that an additional mechanism is at play.

Spatial extent of a single lipid's influence on bilayer mechanics.

To what spatial extent does a single lipid affect the mechanical properties of the membrane that surrounds it? The lipid composition of a membrane determines its mechanical properties. The shapes available to the membrane depend on its compositional material properties, and therefore, the lipid environment. Because each individual lipid species' chemistry is different, it is important to know its range of influence on membrane mechanical properties. This is defined herein as the lipid's mechanical extent. Here, a lipid's mechanical extent is determined by quantifying lipid redistribution and the average curvature that lipid species experience on fluctuating membrane surfaces. A surprising finding is that, unlike unsaturated lipids, saturated lipids have a complicated, nonlocal effect on the surrounding surface, with the interaction strength maximal at a finite length-scale. The methodology provides the means to substantially enrich curvature-energy models of membrane structures, quantifying what was previously only conjecture.

Curvature Energetics Determined by Alchemical Simulation on Four Topologically Distinct Lipid Phases.

The relative curvature energetics of two lipids are tested using thermodynamic integration (TI) on four topologically distinct lipid phases. Simulations use TI to switch between choline headgroup lipids (POPC; that prefers to be flat) and ethanolamine headgroup lipids (POPE; that prefer, for example, the inner monolayer of vesicles). The thermodynamical moving of the lipids between planar, inverse hexagonal (HII), cubic (QII; Pn3m space group), and vesicle topologies reveals differences in material parameters that were previously challenging to access. The methodology allows for predictions of two important lipid material properties: the difference in POPC/POPE monolayer intrinsic curvature (ΔJ0) and the difference in POPC/POPE monolayer Gaussian curvature modulus (Δκ̅m), both of which are connected to the energetics of topological variation. Analysis of the TI data indicates that, consistent with previous experiment and simulation, the J0 of POPE is more negative than POPC (ΔJ0 = -0.018 ± 0.001 Å-1). The theoretical framework extracts significant differences in κ̅m of which POPE is less negative than POPC by 2.0 to 4.0 kcal/mol. The range of these values is determined by considering subsets of the simulations, and disagreement between these subsets suggests separate mechanical parameters at very high curvature. Finally, the fit of the TI data to the model indicates that the position of the pivotal plane of curvature is not constant across topologies at high curvature. Overall, the results offer insights into lipid material properties, the limits of a single HC model, and how to test them using simulation.

A combined molecular/continuum-modeling approach to predict the small-angle neutron scattering of curved membranes.

This paper develops a framework to compute the small-angle neutron scattering (SANS) from highly curved, dynamically fluctuating, and potentially inhomogeneous membranes. This method is needed to compute the scattering from nanometer-scale membrane domains that couple to curvature, as predicted by molecular modeling. The detailed neutron scattering length density of a small planar bilayer patch is readily available via molecular dynamics simulation. A mathematical, mechanical transformation of the planar scattering length density is developed to predict the scattering from curved bilayers. By simulating a fluctuating, curved, surface-continuum model, long time- and length-scales can be reached while, with the aid of the planar-to-curved transformation, the molecular features of the scattering length density can be retained. A test case for the method is developed by constructing a coarse-grained lipid vesicle following a protocol designed to relieve both the osmotic stress inside the vesicle and the lipid-number stress between the leaflets. A question was whether the hybrid model would be able to replicate the scattering from the highly deformed inner and outer leaflets of the small vesicle. Matching the scattering of the full (molecular vesicle) and hybrid (continuum vesicle) models indicated that the inner and outer leaflets of the full vesicle were expanded laterally, consistent with previous simulations of the Martini forcefield that showed thinning in small vesicles. The vesicle structure is inconsistent with a zero-tension leaflet deformed by a single set of elastic parameters, and the results show that this is evident in the scattering. The method can be applied to translate observations of any molecular model's neutron scattering length densities from small patches to large length and timescales.

Laterally Resolved Small-Angle Scattering Intensity from Lipid Bilayer Simulations: An Exact and a Limited-Range Treatment.

When combined, molecular simulations and small-angle scattering experiments are able to provide molecular-scale resolution of structure. Separately, scattering experiments provide only intermingled pair correlations between atoms, while molecular simulations are limited by model quality and the relatively short time scales that they can access. Their combined strength relies on agreement between the experimental spectra and those computed by simulation. To date, computing the neutron spectra from a molecular simulation of a lipid bilayer is straightforward only if the structure is approximated by laterally averaging the in-plane bilayer structure. However, this neglects all information about lateral heterogeneity, e.g., clustering of components in a lipid mixture. This paper presents two methods for computing the scattering intensity of simulated bilayers with in-plane heterogeneity, enabling a full treatment of both the transverse and lateral bilayer structure for the first time. The first method, termed the Dirac Brush, computes the exact spectra including spurious artifacts resulting from using information from neighboring periodic cells to account for the long-range structure of the bilayer. The second method, termed PFFT, applies a mean-field treatment in the field far from a scattering element, resulting in a correlation range that can be tuned (eliminating correlations with neighboring periodic images), but with computational cost that prohibits obtaining the exact (Dirac Brush) spectra. Following their derivation, the two methods are applied to a coarse-grained molecular simulation of a bilayer inhomogeneity, demonstrating the contributions of lateral correlations to the resulting spectra.

An implicit lipid model for efficient reaction-diffusion simulations of protein binding to surfaces of arbitrary topology.

Localization of proteins to a membrane is an essential step in a broad range of biological processes such as signaling, virion formation, and clathrin-mediated endocytosis. The strength and specificity of proteins binding to a membrane depend on the lipid composition. Single-particle reaction-diffusion methods offer a powerful tool for capturing lipid-specific binding to membrane surfaces by treating lipids explicitly as individual diffusible binding sites. However, modeling lipid particle populations is expensive. Here, we present an algorithm for reversible binding of proteins to continuum surfaces with implicit lipids, providing dramatic speed-ups to many body simulations. Our algorithm can be readily integrated into most reaction-diffusion software packages. We characterize changes to kinetics that emerge from explicit vs implicit lipids as well as surface adsorption models, showing excellent agreement between our method and the full explicit lipid model. Compared to models of surface adsorption, which couple together binding affinity and lipid concentration, our implicit lipid model decouples them to provide more flexibility for controlling surface binding properties and lipid inhomogeneity, thus reproducing binding kinetics and equilibria. Crucially, we demonstrate our method's application to membranes of arbitrary curvature and topology, modeled via a subdivision limit surface, again showing excellent agreement with explicit lipid simulations. Unlike adsorption models, our method retains the ability to bind lipids after proteins are localized to the surface (through, e.g., a protein-protein interaction), which can greatly increase the stability of multiprotein complexes on the surface. Our method will enable efficient cell-scale simulations involving proteins localizing to realistic membrane models, which is a critical step for predictive modeling and quantification of in vitro and in vivo dynamics.

Simulation Best Practices for Lipid Membranes [Article v1.0].

We establish a reliable and robust standardization of settings for practical molecular dynamics (MD) simulations of pure and mixed (single- and multi-component) lipid bilayer membranes. In lipid membranes research, particle-based molecular simulations are a powerful tool alongside continuum theory, lipidomics, and model, in vitro, and in vivo experiments. Molecular simulations can provide precise and reproducible spatiotemporal (atomic- and femtosecond-level) information about membrane structure, mechanics, thermodynamics, kinetics, and dynamics. Yet the simulation of lipid membranes can be a daunting task, given the uniqueness of lipid membranes relative to conventional liquid-liquid and solid-liquid interfaces, the immense and complex thermodynamic and statistical mechanical theory, the diversity of multiscale lipid models, limitations of modern computing power, the difficulty and ambiguity of simulation controls, finite size effects, competitive continuum simulation alternatives, and the desired application, including vesicle experiments and biological membranes. These issues can complicate an essential understanding of the field of lipid membranes, and create major bottlenecks to simulation advancement. In this article, we clarify these issues and present a consistent, thorough, and user-friendly framework for the design of state-of-the-art lipid membrane MD simulations. We hope to allow early-career researchers to quickly overcome common obstacles in the field of lipid membranes and reach maximal impact in their simulations.

Building Blocks of the Outer Membrane: Calculating a General Elastic Energy Model for ß-Barrel Membrane Proteins.

The outer membranes of Gram negative bacteria are the first points of contact these organisms make with their environment. Understanding how composition determines the mechanical properties of this essential barrier is of paramount importance. Therefore, we developed a new computational method to measure the elasticity of transmembrane proteins found in the outer membrane. Using all-atom molecular dynamics simulations of these proteins, we apply a set of external forces to mechanically stress the transmembrane ß-barrels. Our results from four representative ß-barrels show that outer membrane proteins display elastic properties that are approximately 70 to 190 times stiffer than neat lipid membranes. These findings suggest that outer membrane ß-barrels are a significant source of mechanical stability in bacteria. Our all-atom approach further reveals that resistance to radial stress is encoded by a general mechanism that includes stretching of backbone hydrogen bonds and tilting of ß-strands with respect to the bilayer normal. This computational framework facilitates an increased theoretical understanding of how varying lipid and protein amounts affect the mechanical properties of the bacterial outer membrane.