Transmembrane ß-Barrel Models of α-Synuclein Oligomers.

The aggregation of α-synuclein is implicated in a number of neurodegenerative diseases, such as Parkinson's and Multiple System Atrophy, but the role of these aggregates in disease development is not clear. One possible mechanism of cytotoxicity is the disturbance or permeabilization of cell membranes by certain types of oligomers. However, no high-resolution structure of such membrane-embedded complexes has ever been determined. Here we construct and evaluate putative transmembrane ß-barrels formed by this protein. Examination of the α-synuclein sequence reveals two regions that could form membrane-embedded ß-hairpins: 64-92 (the NAC), and 35-56, which harbors many familial Parkinson's mutations. The stability of ß-barrels formed by these hairpins is examined first in implicit membrane pores and then by multimicrosecond all-atom simulations. We find that a NAC region barrel remains stably inserted and hydrated for at least 10 µs. A 35-56 barrel remains stably inserted in the membrane but dehydrates and collapses if all His50 are neutral or if His50 is replaced by Q. If half of the His50 are doubly protonated, the barrel takes an oval shape but remains hydrated for at least 10 µs. Possible implications of these findings for α-synuclein pathology are discussed.

Comparison of classical and ab initio simulations of hydronium and aqueous proton transfer.

Proton transport in aqueous systems occurs by making and breaking covalent bonds, a process that classical force fields cannot reproduce. Various attempts have been made to remedy this deficiency, by valence bond theory or instantaneous proton transfers, but the ability of such methods to provide a realistic picture of this fundamental process has not been fully evaluated. Here we compare an ab initio molecular dynamics (AIMD) simulation of an excess proton in water to a simulation of a classical H3O+ in TIP3P water. The energy gap upon instantaneous proton transfer from H3O+ to an acceptor water molecule is much higher in the classical simulation than in the AIMD configurations evaluated with the same classical potential. The origins of this discrepancy are identified by comparing the solvent structures around the excess proton in the two systems. One major structural difference is in the tilt angle of the water molecules that accept an hydrogen bond from H3O+. The lack of lone pairs in TIP3P produces a tilt angle that is too large and generates an unfavorable geometry after instantaneous proton transfer. This problem can be alleviated by the use of TIP5P, which gives a tilt angle much closer to the AIMD result. Another important factor that raises the energy gap is the different optimal distance in water-water vs H3O+-water H-bonds. In AIMD the acceptor is gradually polarized and takes a hydronium-like configuration even before proton transfer actually happens. Ways to remedy some of these problems in classical simulations are discussed.

Proton Paths in Models of the Hv1 Proton Channel.

The voltage-gated proton channel (Hv1) plays an essential role in numerous biological processes, but a detailed molecular understanding of its function is lacking. The lack of reliable structures for the open and resting states is a major handicap. Several models have been built based on homologous voltage sensors and the structure of a chimera between the mouse homologue and a phosphatase voltage sensor, but their validity is uncertain. In addition, differing views exist regarding the mode of proton translocation, the role of specific residues, and the mechanism of pH effects on voltage gating. Here we use classical proton hopping simulations under a voltage biasing force to evaluate some of the proposed structural models and explore the mechanism of proton conduction. Paradoxically, some models proposed for the closed state allow for proton permeation more easily than models for the open state. An open state model with a D112-R211 salt bridge (R3D) allows proton transport more easily than models with a D112-R208 salt bridge (R2D). However, its permeation rate seems too high, considering experimental conductances. In all cases, the proton permeates through a water wire, bypassing the salt-bridged D112 rather than being shuttled by D112. Attempts to protonate D112 are rejected due to its strong interaction with an arginine. Consistent with proton selectivity, no Na+ permeation was observed in the R2D models. As a negative control, simulations with the Kv1.2-Kv2.1 paddle-chimera voltage sensor, which is not expected to conduct protons, did not show proton permeation under the same conditions. Hydrogen bond connectivity graphs show a constriction at D112, but cannot discriminate between open and closed states.

Simulations suggest a scaffolding mechanism of membrane deformation by the caveolin 8S complex.

Caveolins form complexes of various sizes that deform membranes into polyhedral shapes. However, the recent structure of the 8S complex was disk-like with a flat membrane-binding surface. How can a flat complex deform membranes into nonplanar structures? Molecular dynamics simulations revealed that the 8S complex rapidly takes the form of a suction cup. Simulations on implicit membrane vesicles determined that binding is stronger when E140 gets protonated. In that case, the complex binds much more strongly to 5- and 10-nm-radius vesicles. A concave membrane-binding surface readily explains the membrane-deforming ability of caveolins by direct scaffolding. We propose that the 8S complex sits at the vertices of the caveolar polyhedra, rather than at the center of the polyhedral faces.

Putative Pore Structures of Amyloid ß 25-35 in Lipid Bilayers.

The amyloid ß peptide aggregates to form extracellular plaques in the brains of Alzheimer's disease patients. Certain of its fragments have been found to have similar properties to those of the full-length peptide. The best-studied of these is 25-35, which aggregates into fibrils, is toxic to neurons, and forms ion channels in synthetic lipid bilayers. Here, we investigate possible pore-forming structures of oligomers of this peptide in a POPC/POPG membrane. We consider octameric and decameric ß-barrels of different topology, strand orientation, and shear, evaluate their stability in an implicit membrane model, and subject the best models to multimicrosecond all-atom molecular dynamics simulations. We find two decameric structures that are kinetically stable in membranes on this time scale: an imperfectly closed antiparallel ß-barrel with K28 in the pore lumen and a short parallel ß-barrel with K28 toward the membrane interface. Both structures exhibit dehydrated gaps in the pore lumen, which are larger for the antiparallel barrel. Based on these results, the experimental cation selectivity, the dependence of ion channel activity on voltage direction, and certain mutation data, the parallel model seems more compatible with experimental data.

Putative Structures of Membrane-Embedded Amyloid ß Oligomers.

Perturbation of cell membranes by amyloid ß (Ab) peptide oligomers is one possible mechanism of cytotoxicity in Alzheimer's disease, but the structure of such Ab-membrane complexes is unknown. Here we examine the stability of several putative structures by implicit membrane and all-atom molecular dynamics simulations. The structures include (a) a variety of models proposed by other researchers in the past, (b) a heptameric ß barrel determined by grafting the Ab sequence onto α-hemolysin, (c) a similar structure with modified strand orientation and turn location based on an experimental ß-hairpin structure, (d) oligomers inserting C-terminal ß hairpins into one leaflet of the bilayer, (e) oligomers forming parallel C-terminal ß barrels, and (f) a helical hexamer made of C-terminal fragments. The α-hemolysin-grafted structure and its alternately oriented variant are stable in the membrane and form an aqueous pore. In contrast, the C-terminal parallel barrels are not stable, presumably due to excessive hydrophobicity of their inner surface. The helical hexamer also failed to stabilize an aqueous pore for the same reason. The C-terminal hairpin-inserting structures remain stably inserted but, again, do not form an aqueous pore. Our results suggest that only ß-barrels inserting a combination of C-terminal and other residues can form stable aqueous pores.

Molecular origins of asymmetric proton conduction in the influenza M2 channel.

The M2 proton channel of influenza A is embedded into the viral envelope and allows acidification of the virion when the external pH is lowered. In contrast, no outward proton conductance is observed when the internal pH is lowered, although outward current is observed at positive voltage. Residues Trp41 and Asp44 are known to play a role in preventing pH-driven outward conductance, but the mechanism for this is unclear. We investigate this issue using classical molecular dynamics simulations with periodic proton hops. When all key His37 residues are neutral, inward proton movement is much more facile than outward movement if the His are allowed to shuttle the proton. The preference for inward movement increases further as the charge on the His37 increases. Analysis of the trajectories reveals three factors accounting for this asymmetry. First, in the outward direction, Asp44 traps the hydronium by strong electrostatic interactions. Secondly, Asp44 and Trp41 orient the hydronium with the protons pointing inward, hampering outward Grotthus hopping. As a result, the effective barrier is lower in the inward direction. Trp41 adds to the barrier by weakly H-bonding to potential H+ acceptors. Finally, for charged His, the H3O+ in the inner vestibule tends to get trapped at lipid-lined fenestrations of the cone-shaped channel. Simulations qualitatively reproduce the experimentally observed higher outward conductance of mutants. The ability of positive voltage, unlike proton gradient, to induce an outward current appears to arise from its ability to bias H3O+ and the waters around it toward more H-outward orientations.

Amino acid deprotonation rates from classical force fields.

Acid ionization constants (pKa's) of titratable amino acid side chains have received a large amount of experimental and theoretical attention. In many situations, however, the rates of protonation and deprotonation, kon and koff, may also be important, for example, in understanding the mechanism of action of proton channels or membrane proteins that couple proton transport to other processes. Protonation and deprotonation involve the making and breaking of covalent bonds, which cannot be studied by classical force fields. However, environment effects on the rates should be captured by such methods. Here, we present an approach for estimating deprotonation rates based on Warshel's extension of Marcus's theory of electron transfer, with input from molecular simulations. The missing bond dissociation energy is represented by a constant term determined by fitting the pKa value in solution. The statistics of the energy gap between protonated and deprotonated states is used to compute free energy curves of the two states and, thus, free energy barriers, from which the rate can be deduced. The method is applied to Glu, Asp, and His in bulk solution and select membrane proteins: the M2 proton channel, bacteriorhodopsin, and cytochrome c oxidase.

Distinct Modes of Action of IAPP Oligomers on Membranes.

Islet amyloid polypeptide (IAPP, also known as amylin) is a peptide hormone that is co-secreted with insulin by pancreatic ß-cells and forms amyloid aggregates in type II diabetes. Various lines of evidence indicate that oligomers of this peptide may induce toxicity by disrupting or forming pores in cell membranes, but the structure of these pores is unknown. Here, we create models of pores for both helical and ß-structured peptides using implicit membrane modeling and test their stability using multimicrosecond all-atom simulations. We find that the helical peptides behave similarly to antimicrobial peptides; they remain stably inserted in a highly tilted or partially unfolded configuration creating a narrow water channel. Parallel helix orientation creates a somewhat larger pore. An octameric ß barrel of parallel ß-hairpins is highly stable in the membrane, whereas the corresponding barrel made of antiparallel hairpins is not. We propose that certain experiments probe the helical pore state while others probe the ß-structured pore state; this provides a possible explanation for lack of correlation that is sometimes observed between in vivo toxicity and in vitro liposome permeabilization experiments.

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 are especially mysterious since 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 give similar results, whereas a flat F-BAR sheet develops 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.

Lipid interactions of an actinoporin pore-forming oligomer.

The actinoporins are cytolytic toxins produced by sea anemones. Upon encountering a membrane, preferably containing sphingomyelin, they oligomerize and insert their N-terminal helix into the membrane, forming a pore. Whether sphingomyelin is specifically recognized by the protein or simply induces phase coexistence in the membrane has been debated. Here, we perform multi-microsecond molecular dynamics simulations of an octamer of fragaceatoxin C, a member of the actinoporin family, in lipid bilayers containing either pure 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or a 1:1 mixture of DOPC and palmitoyl sphingomyelin (PSM). The complex is highly stable in both environments, with only slight fraying of the inserted helices near their N-termini. Analyzing the structural parameters of the mixed membrane in the course of the simulation, we see signs of a phase transition for PSM in the inner leaflet of the bilayer. In both leaflets, cross-interactions between lipids of different type decrease over time. Surprisingly, the aromatic loop thought to be responsible for sphingomyelin recognition interacts more with DOPC than PSM by the end of the simulation. These results support the notion that the key membrane property that actinoporins recognize is lipid phase coexistence.

Free energy of hydrophilic and hydrophobic pores in lipid bilayers by free energy perturbation of a restraint.

The free energy of pore formation in lipid bilayers has been previously calculated using a variety of reaction coordinates. Here, we use free energy perturbation of a cylindrical lipid exclusion restraint to compute the free energy profile as a function of pore radius in dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC) bilayers. Additionally restraining the headgroups to lie on the membrane surface allows us to also calculate the free energy profile of hydrophobic pores, i.e., cylindrical pores lined by acyl chains. For certain pore radii, the free energy of wetting of hydrophobic pores is calculated using the density bias method. It is found that wetting of hydrophobic pores becomes thermodynamically favorable at 5.0 Å for DMPC and 6.5 Å for DOPC, although significant barriers prevent spontaneous wetting of the latter on a nanosecond time scale. The free energy of transformation of hydrophilic pores to hydrophobic ones is also calculated using free energy perturbation of headgroup restraints along the bilayer normal. This quantity, along with wetting and pore growth free energies, provides complete free energy profiles as a function of radius. Pore line tension values for the hydrophilic pores obtained from the slope of the free energy profiles are 37.6 pN for DMPC and 53.7 pN for DOPC. The free energy profiles for the hydrophobic pores are analyzed in terms of elementary interfacial tensions. It is found that a positive three-phase line tension is required to explain the results. The estimated value for this three-phase line tension (51.2 pN) lies within the expected range.

Correction to: Experimental and Computational Characterization of Oxidized and Reduced Protegrin Pores in Lipid Bilayers.

The original version of the article was published without the Graphic Abstract. Graphic Abstract image of the article is given below.

The value of antimicrobial peptides in the age of resistance.

Accelerating growth and global expansion of antimicrobial resistance has deepened the need for discovery of novel antimicrobial agents. Antimicrobial peptides have clear advantages over conventional antibiotics which include slower emergence of resistance, broad-spectrum antibiofilm activity, and the ability to favourably modulate the host immune response. Broad bacterial susceptibility to antimicrobial peptides offers an additional tool to expand knowledge about the evolution of antimicrobial resistance. Structural and functional limitations, combined with a stricter regulatory environment, have hampered the clinical translation of antimicrobial peptides as potential therapeutic agents. Existing computational and experimental tools attempt to ease the preclinical and clinical development of antimicrobial peptides as novel therapeutics. This Review identifies the benefits, challenges, and opportunities of using antimicrobial peptides against multidrug-resistant pathogens, highlights advances in the deployment of novel promising antimicrobial peptides, and underlines the needs and priorities in designing focused development strategies taking into account the most advanced tools available.

Experimental and Computational Characterization of Oxidized and Reduced Protegrin Pores in Lipid Bilayers.

Protegrin-1 (PG-1), an 18-residue ß-hairpin stabilized by two disulfide bonds, is a member of a family of powerful antimicrobial peptides which are believed to act through membrane permeabilization. Here we used a combination of experimental and computational approaches to characterize possible structural arrangements of PG-1 in lipid bilayers mimicking bacterial membranes. We have measured the dose-response function of the PG-1-induced leakage of markers of various sizes from vesicles and found it to be consistent with the formation of pores of two different sizes. The first one allows the release of small dyes and occurs at peptide:lipid ratios < 0.006. Above this ratio, larger pores are observed through which the smallest of dextrans FD4 can be released. In parallel with pore formation, we observe a general large-scale destabilization of vesicles which is probably related to complete rupture of some vesicles. The population of vesicles that are completely ruptured depends linearly on PG-1:lipid ratio. Neither pore size, nor vesicle rupture are influenced by the formation of disulfide bonds. Previous computational work on oxidized protegrin is complemented here by all-atom MD simulations of PG-1 with reduced disulfide bonds both in solution (monomer) and in a bilayer (dimer and octamer). The simulations provide molecular insights into the influence of disulfide bonds on peptide conformation, aggregation, and oligomeric structure.

Mechanisms of negative membrane curvature sensing and generation by ESCRT III subunit Snf7.

Certain proteins have the propensity to bind to negatively curved membranes and generate negative membrane curvature. The mechanism of action of these proteins is much less studied and understood than those that sense and generate positive curvature. In this work, we use implicit membrane modeling to explore the mechanism of an important negative curvature sensing and generating protein: the main ESCRT III subunit Snf7. We find that Snf7 monomers alone can sense negative curvature and that curvature sensitivity increases for dimers and trimers. We have observed spontaneous bending of Snf7 oligomers into circular structures with preferred radius of ~20 nm. The preferred curvature of Snf7 filaments is further confirmed by the simulations of preformed spirals on a cylindrical membrane surface. Snf7 filaments cannot bind with the same interface to flat and curved membranes. We find that even when a filament has the preferred radius, it is always less stable on the flat membrane surface than on the interior cylindrical membrane surface. This provides an additional energy for membrane bending which has not been considered in the spiral spring model. Furthermore, the rings on the cylindrical spirals are bridged together by helix 4 and hence are extra stabilized compared to the spirals on the flat membrane surface.

What Makes a Good Pore Former: A Study of Synthetic Melittin Derivatives.

Pore formation by membrane-active peptides, naturally encountered in innate immunity and infection, could have important medical and technological applications. Recently, the well-studied lytic peptide melittin has formed the basis for the development of combinatorial libraries from which potent pore-forming peptides have been derived, optimized to work under different conditions. We investigate three such peptides, macrolittin70, which is most active at neutral pH; pHD15, which is active only at low pH; and MelP5_Δ6, which was rationally designed to be active at low pH but formed only small pores. There are three, six, and six acidic residues in macrolittin70, pHD15, and MelP5_Δ6, respectively. We perform multi-microsecond simulations in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) of hexamers of these peptides starting from transmembrane orientations at neutral pH (all residues at standard protonation), low pH (acidic residues and His protonated), and highly acidic environments in which C-termini are also protonated. Previous simulations of the parent peptides melittin and MelP5 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are repeated in POPC. We find that the most potent pore-forming peptides exhibit strong interpeptide interactions, including salt bridges, H-bonds, and polar interactions. Protonation of the C-terminus promotes helicity and pore size. The proximity of the peptides allows fewer lipid headgroups to line the pores than in previous simulations, making the pores intermediate between barrel stave and toroidal. Based on these structures and geometrical arguments, we attempt to rationalize the factors that under different conditions can increase or decrease pore stability and propose mutations that could be tested experimentally.

Effects of Peptide Charge, Orientation, and Concentration on Melittin Transmembrane Pores.

Melittin is a short cationic peptide that exerts cytolytic effects on bacterial and eukaryotic cells. Experiments suggest that in zwitterionic membranes, melittin forms transmembrane toroidal pores supported by four to eight peptides. A recently constructed melittin variant with a reduced cationic charge, MelP5, is active at 10-fold lower concentrations. In previous work, we performed molecular dynamics simulations on the microsecond timescale to examine the supramolecular pore structure of a melittin tetramer in zwitterionic and partially anionic membranes. We now extend that study to include the effects of peptide charge, initial orientation, and number of monomers on the pore formation and stabilization processes. Our results show that parallel transmembrane orientations of melittin and MelP5 are more consistent with experimental data. Whereas a MelP5 parallel hexamer forms a large stable pore during the 5-µs simulation time, a melittin hexamer and an octamer are not fully stable, with several monomers dissociating during the simulation time. Interaction-energy analysis shows that this difference in behavior between melittin and MelP5 is not due to stronger electrostatic repulsion between neighboring melittin peptides but to peptide-lipid interactions that disfavor the isolated MelP5 transmembrane monomer. The ability of melittin monomers to diffuse freely in the 1,2-dimyristoyl-SN-glycero-3-phosphocholine membrane leads to dynamic pores with varying molecularity.

Membrane Curvature Sensing by Amphipathic Helices: Insights from Implicit Membrane Modeling.

Sensing and generation of lipid membrane curvature, mediated by the binding of specific proteins onto the membrane surface, play crucial roles in cell biology. A number of mechanisms have been proposed, but the molecular understanding of these processes is incomplete. All-atom molecular dynamics simulations have offered valuable insights but are extremely demanding computationally. Implicit membrane simulations could provide a viable alternative, but current models apply only to planar membranes. In this work, the implicit membrane model 1 is extended to spherical and tubular membranes. The geometric change from planar to curved shapes is straightforward but insufficient for capturing the full curvature effect, which includes changes in lipid packing. Here, these packing effects are taken into account via the lateral pressure profile. The extended implicit membrane model 1 is tested on the wild-types and mutants of the antimicrobial peptide magainin, the ALPS motif of arfgap1, α-synuclein, and an ENTH domain. In these systems, the model is in qualitative agreement with experiments. We confirm that favorable electrostatic interactions tend to weaken curvature sensitivity in the presence of strong hydrophobic interactions but may actually have a positive effect when those are weak. We also find that binding to vesicles is more favorable than binding to tubes of the same diameter and that the long helix of α-synuclein tends to orient along the axis of tubes, whereas shorter helices tend to orient perpendicular to it. Adoption of a specific orientation could provide a mechanism for coupling protein oligomerization to tubule formation.

Classical Molecular Dynamics with Mobile Protons.

An important limitation of standard classical molecular dynamics simulations is the inability to make or break chemical bonds. This restricts severely our ability to study processes that involve even the simplest of chemical reactions, the transfer of a proton. Existing approaches for allowing proton transfer in the context of classical mechanics are rather cumbersome and have not achieved widespread use and routine status. Here we reconsider the combination of molecular dynamics with periodic stochastic proton hops. To ensure computational efficiency, we propose a non-Boltzmann acceptance criterion that is heuristically adjusted to maintain the correct or desirable thermodynamic equilibria between different protonation states and proton transfer rates. Parameters are proposed for hydronium, Asp, Glu, and His. The algorithm is implemented in the program CHARMM and tested on proton diffusion in bulk water and carbon nanotubes and on proton conductance in the gramicidin A channel. Using hopping parameters determined from proton diffusion in bulk water, the model reproduces the enhanced proton diffusivity in carbon nanotubes and gives a reasonable estimate of the proton conductance in gramicidin A.