How proteins open fusion pores: insights from molecular simulations.

Fusion proteins can play a versatile and involved role during all stages of the fusion reaction. Their roles go far beyond forcing the opposing membranes into close proximity to drive stalk formation and fusion. Molecular simulations have played a central role in providing a molecular understanding of how fusion proteins actively overcome the free energy barriers of the fusion reaction up to the expansion of the fusion pore. Unexpectedly, molecular simulations have revealed a preference of the biological fusion reaction to proceed through asymmetric pathways resulting in the formation of, e.g., a stalk-hole complex, rim-pore, or vertex pore. Force-field based molecular simulations are now able to directly resolve the minimum free-energy path in protein-mediated fusion as well as quantifying the free energies of formed reaction intermediates. Ongoing developments in Graphics Processing Units (GPUs), free energy calculations, and coarse-grained force-fields will soon gain additional insights into the diverse roles of fusion proteins.

Liquids relax and unify strain in graphene.

Solid substrates often induce non-uniform strain and doping in graphene monolayer, therefore altering the intrinsic properties of graphene, reducing its charge carrier mobilities and, consequently, the overall electrical performance. Here, we exploit confocal Raman spectroscopy to study graphene directly free-floating on the surface of water, and show that liquid supports relief the preexisting strain, have negligible doping effect and restore the uniformity of the properties throughout the graphene sheet. Such an effect originates from the structural adaptability and flexibility, lesser contamination and weaker intermolecular bonding of liquids compared to solid supports, independently of the chemical nature of the liquid. Moreover, we demonstrate that water provides a platform to study and distinguish chemical defects from substrate-induced defects, in the particular case of hydrogenated graphene. Liquid supports, thus, are advantageous over solid supports for a range of applications, particularly for monitoring changes in the graphene structure upon chemical modification.

Fusion Pores Live on the Edge.

Biological transmission of vesicular content occurs by opening of a fusion pore. Recent experimental observations have illustrated that fusion pores between vesicles that are docked by an extended flat contact zone are located at the edge (vertex) of this zone. We modeled this experimentally observed scenario by coarse-grained molecular simulations and elastic theory. This revealed that fusion pores experience a direct attraction toward the vertex. The size adopted by the resulting vertex pore strongly depends on the apparent contact angle between the adhered vesicles even in the absence of membrane surface tension. Larger contact angles substantially increase the equilibrium size of the vertex pore. Because the cellular membrane fusion machinery actively docks membranes, it facilitates a collective expansion of the contact zone and increases the contact angle. In this way, the fusion machinery can drive expansion of the fusion pore by free energy equivalents of multiple tens of kBT from a distance and not only through the fusion proteins that reside within the fusion pore.

The 2018 biomembrane curvature and remodeling roadmap.

The importance of curvature as a structural feature of biological membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one hand, changes in membrane morphology are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo- and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of Journal of Physics D: Applied Physics addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area.

PspF-binding domain PspA1-144 and the PspA·F complex: New insights into the coiled-coil-dependent regulation of AAA+ proteins.

Phage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ(54) enhancer-binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ(54) -dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiled-coil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiologic tasks via coiled-coil domains.

TatBC-independent TatA/Tat substrate interactions contribute to transport efficiency.

The Tat system can transport folded, signal peptide-containing proteins (Tat substrates) across energized membranes of prokaryotes and plant plastids. A twin-arginine motif in the signal peptide of Tat substrates is recognized by TatC-containing complexes, and TatA permits the membrane passage. Often, as in the model Tat systems of Escherichia coli and plant plastids, a third component - TatB - is involved that resembles TatA but has a higher affinity to TatC. It is not known why most TatA dissociates from TatBC complexes in vivo and distributes more evenly in the membrane. Here we show a TatBC-independent substrate-binding to TatA from Escherichia coli, and we provide evidence that this binding enhances Tat transport. First hints came from in vivo cross-linking data, which could be confirmed by affinity co-purification of TatA with the natural Tat substrates HiPIP and NrfC. Two positions on the surface of HiPIP could be identified that are important for the TatA interaction and transport efficiency, indicating physiological relevance of the interaction. Distributed TatA thus may serve to accompany membrane-interacting Tat substrates to the few TatBC spots in the cells.

Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment.

Synaptic-vesicle exocytosis is mediated by the vesicular Ca(2+) sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca(2+) through PIP2. This interaction allows both Ca(2+)-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca(2+) triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca(2+) influx bringing the vesicle membrane close enough for membrane fusion.

Membrane lateral structure: the influence of immobilized particles on domain size.

In experiments on model membranes, formation of large domains of different lipid composition is readily observed. However, no such phase separation is observed in the membranes of intact cells. Instead, small transient inhomogeneities called lipid rafts are expected in these systems. One of the numerous attempts to explain small domains refers to the coupling of the membrane to its surroundings, which leads to the immobilization of some of the membrane molecules. These immobilized molecules then act as static obstacles for the remaining mobile ones. We present detailed Molecular Dynamics simulations demonstrating that this can indeed account for small domains. This confirms previous Monte Carlo studies based on simplified models. Furthermore, by directly comparing domain structures obtained using Molecular Dynamics to Monte Carlo simulations of the Ising model, we demonstrate that domain formation in the presence of obstacles is remarkably insensitive to the details of the molecular interactions.

Membrane protein sequestering by ionic protein-lipid interactions.

Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis. However, how syntaxin-1A is sequestered is unknown. Here we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using super-resolution stimulated-emission depletion microscopy on the plasma membranes of PC12 cells, we found that PIP2 is the dominant inner-leaflet lipid in microdomains about 73 nanometres in size. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the carboxy-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independently of cholesterol or lipid phases.

Curvature-dependent elastic properties of liquid-ordered domains result in inverted domain sorting on uniaxially compressed vesicles.

Using a coarse-grained molecular model we study the spatial distribution of lipid domains on a 20-nm-sized vesicle. The lipid mixture laterally phase separates into a raftlike, liquid-ordered (l(o)) phase and a liquid-disordered phase. As we uniaxially compress the mixed vesicle keeping the enclosed volume constant, we impart tension onto the membrane. The vesicle adopts a barrel shape, which is composed of two flat contact zones and a curved edge. The l(o) domain, which exhibits a higher bending rigidity, segregates to the highly curved edge. This inverted domain sorting switches to normal domain sorting, where the l(o) domain prefers the flat contact zone, when we release the contents of the vesicle. We rationalize this domain sorting by a pronounced reduction of the bending rigidity and area compressibility of the l(o) phase upon bending.

Release of content through mechano-sensitive gates in pressurized liposomes.

Mechano-sensitive channels are ubiquitous membrane proteins that activate in response to increasing tension in the lipid membrane. They facilitate a sudden, nonselective release of solutes and water that safeguards the integrity of the cell in hypo- or hyper-osmotic shock conditions. We have simulated the rapid release of content from a pressurized liposome through a particular mechano-sensitive protein channel, MscL, embedded in the liposomal membrane. We show that a single channel is able to relax the liposome, stressed to the point of bursting, in a matter of microseconds. We map the full activation-deactivation cycle of MscL in near-atomic detail and are able to quantify the rapid decrease in liposomal stress as a result of channel activation. This provides a computational tool that opens the way to contribute to the rational design of functional nano-containers.

Reconstruction of atomistic details from coarse-grained structures.

We present an algorithm to reconstruct atomistic structures from their corresponding coarse-grained (CG) representations and its implementation into the freely available molecular dynamics (MD) program package GROMACS. The central part of the algorithm is a simulated annealing MD simulation in which the CG and atomistic structures are coupled via restraints. A number of examples demonstrate the application of the reconstruction procedure to obtain low-energy atomistic structural ensembles from their CG counterparts. We reconstructed individual molecules in vacuo (NCQ tripeptide, dipalmitoylphosphatidylcholine, and cholesterol), bulk water, and a WALP transmembrane peptide embedded in a solvated lipid bilayer. The first examples serve to optimize the parameters for the reconstruction procedure, whereas the latter examples illustrate the applicability to condensed-phase biomolecular systems.

Stability of asymmetric lipid bilayers assessed by molecular dynamics simulations.

The asymmetric insertion of amphiphiles into biological membranes compromises the balance between the inner and outer monolayers. As a result, area expansion of the receiving leaflet and curvature strain may lead to membrane permeation, shape changes, or membrane fusion events. We have conducted both atomistic and coarse-grained molecular dynamics simulations of dipalmitoyl-phosphatidylcholine (DPPC) bilayers to study the effect of an asymmetric distribution of lipids between the two monolayers on membrane stability. Highly asymmetric lipid bilayers were found to be surprisingly stable within the submicrosecond time span of the simulations. Even the limiting case of a monolayer immersed in water ruptured spontaneously only after at least 20 ns simulation. A thermal shock could destabilize these kinetically trapped states. We also studied mixed systems composed of DPPC and short tail diC(8)PC lipids, showing that the presence of the cone-shaped short tail lipid facilitates the release of tension in the asymmetric systems via formation of a transmembrane pore. Thus, asymmetric area expansion and curvature stress cooperate to yield bilayer disruption. It appears that, although asymmetric area expansion destabilizes the bilayer structure, the activation energy for transmonolayer lipid re-equilibration is increased. Such a large kinetic barrier can be reduced by lipids with positive spontaneous curvature. These effects are important at the onset of bilayer destabilization phenomena, such as lipid pore formation and membrane fusion, and should be considered for the mechanism of induction of such processes by peptides and proteins.

3D pressure field in lipid membranes and membrane-protein complexes.

We calculate full 3D pressure fields for inhomogeneous nanoscale systems using molecular dynamics simulation data. The fields represent systems with increasing level of complexity, ranging from semivesicles and vesicles to membranes characterized by coexistence of two phases, including also a protein-membrane complex. We show that the 3D pressure field is distinctly different for curved and planar bilayers, the pressure field depends strongly on the phase of the membrane, and that an integral protein modulates the tension and elastic properties of the membrane.

Curvature effects on lipid packing and dynamics in liposomes revealed by coarse grained molecular dynamics simulations.

The molecular packing details of lipids in planar bilayers are well characterized. For curved bilayers, however, little data is available. In this paper we study the effect of temperature and membrane composition on the structural and dynamical properties of a liposomal membrane in the limit of high curvature (liposomal diameter of 15-20 nm), using coarse grained molecular dynamics simulations. Both pure dipalmitoyl phosphatidylcholine (DPPC) liposomes and binary mixtures of DPPC and either dipalmitoylphosphatidylethanolamine (DPPE) or polyunsaturated dilinoleylphosphatidylcholine (DLiPC) lipids are modeled. We take special care in the equilibration of the liposomes requiring lipid flip-flopping, which can be facilitated by the temporary insertion of artificial pores. The equilibrated liposomes show some remarkable properties. Curvature induces membrane thinning and reduces the thermal expansivity of the membrane. In the inner monolayer the lipid head groups are very closely packed and dehydrated, and the lipids tails relatively disordered. The opposite packing effects are seen in the outer monolayer. In addition, we noticed an increased tendency of the lipid tails to backfold toward the interface in the outer monolayer. The distribution of lipids over the monolayers was found to be strongly temperature dependent. Higher temperatures favor more equally populated monolayers. Relaxation times of the lipid tails were found to increase with increasing curvature, with the lipid tails in the outer monolayer showing a significant slower dynamics compared to the lipid tails in the inner monolayer. In the binary systems there is a clear tendency toward partial transversal demixing of the two components, with especially DPPE enriched in the inner monolayer. This observation is in line with a static shape concept which dictates that inverted-cone shaped lipids such as DPPE and DLiPC would prefer the concave volume of the inner monolayer. However, our results for DLiPC show that another effect comes into play that is almost equally strong and provides a counter-acting driving force toward the outer, rather than the inner monolayer. This effect is the ability of the polyunsaturated tails of DLiPC to backfold, which is advantageous in the outer monolayer. We speculate that polyunsaturated lipids in biological membranes may play an important role in stabilizing both positive and negative regions of curvature.

The molecular face of lipid rafts in model membranes.

Cell membranes contain a large number of different lipid species. Such a multicomponent mixture exhibits a complex phase behavior with regions of structural and compositional heterogeneity. Especially domains formed in ternary mixtures, composed of saturated and unsaturated lipids together with cholesterol, have received a lot of attention as they may resemble raft formation in real cells. Here we apply a simulation model to assess the molecular nature of these domains at the nanoscale, information that has thus far eluded experimental determination. We are able to show the spontaneous separation of a saturated phosphatidylcholine (PC)/unsaturated PC/cholesterol mixture into a liquid-ordered and a liquid-disordered phase with structural and dynamic properties closely matching experimental data. The near-atomic resolution of the simulations reveals remarkable features of both domains and the boundary domain interface. Furthermore, we predict the existence of a small surface tension between the monolayer leaflets that drives registration of the domains. At the level of molecular detail, raft-like lipid mixtures show a surprising face with possible implications for many cell membrane processes.

The molecular mechanism of lipid monolayer collapse.

Lipid monolayers at an air-water interface can be compressed laterally and reach high surface density. Beyond a certain threshold, they become unstable and collapse. Lipid monolayer collapse plays an important role in the regulation of surface tension at the air-liquid interface in the lungs. Although the structures of lipid aggregates formed upon collapse can be characterized experimentally, the mechanism leading to these structures is not fully understood. We investigate the molecular mechanism of monolayer collapse using molecular dynamics simulations. Upon lateral compression, the collapse begins with buckling of the monolayer, followed by folding of the buckle into a bilayer in the water phase. Folding leads to an increase in the monolayer surface tension, which reaches the equilibrium spreading value. Immediately after their formation, the bilayer folds have a flat semielliptical shape, in agreement with theoretical predictions. The folds undergo further transformation and form either flat circular bilayers or vesicles. The transformation pathway depends on macroscopic parameters of the system: the bending modulus, the line tension at the monolayer-bilayer connection, and the line tension at the bilayer perimeter. These parameters are determined by the system composition and temperature. Coexistence of the monolayer with lipid aggregates is favorable at lower tensions of the monolayer-bilayer connection. Transformation into a vesicle reduces the energy of the fold perimeter and is facilitated for softer bilayers, e.g., those with a higher content of unsaturated lipids, or at higher temperatures.

Application of mean field boundary potentials in simulations of lipid vesicles.

A method is presented to enhance the efficiency of simulations of lipid vesicles. The method increases computational speed by eliminating water molecules that either surround the vesicle or reside in the interior of the vesicle, without altering the properties of the water at the membrane interface. Specifically, mean field force approximation (MFFA) boundary potentials are used to replace both the internal and external excess bulk solvent. In addition to reducing the cost of simulating preformed vesicles, the molding effect of the boundary potentials also enhances the formation and equilibration of vesicles from random solutions of lipid in water. Vesicles with diameters in the range from 20 to 60 nm were obtained on a nanosecond time scale, without any noticeable effect of the boundary potentials on their structure.

The MARTINI force field: coarse grained model for biomolecular simulations.

We present an improved and extended version of our coarse grained lipid model. The new version, coined the MARTINI force field, is parametrized in a systematic way, based on the reproduction of partitioning free energies between polar and apolar phases of a large number of chemical compounds. To reproduce the free energies of these chemical building blocks, the number of possible interaction levels of the coarse-grained sites has increased compared to those of the previous model. Application of the new model to lipid bilayers shows an improved behavior in terms of the stress profile across the bilayer and the tendency to form pores. An extension of the force field now also allows the simulation of planar (ring) compounds, including sterols. Application to a bilayer/cholesterol system at various concentrations shows the typical cholesterol condensation effect similar to that observed in all atom representations.