Binding of SV40's Viral Capsid Protein VP1 to Its Glycosphingolipid Receptor GM1 Induces Negative Membrane Curvature: A Molecular Dynamics Study.

The binding of the pentameric capsid protein VP1 of simian virus 40 to its glycosphingolipid receptor GM1 is a key step for the entry of the virus into the host cell. Recent experimental studies have shown that the interaction of variants of soluble VP1 pentamers with giant unilamellar vesicles composed of GM1, DOPC, and cholesterol leads to the formation of tubular membrane invaginations to the inside of the vesicles, mimicking the initial steps of endocytosis. We have used coarse-grained and atomistic molecular dynamics (MD) simulations to study the interaction of VP1 with GM1/DOPC/cholesterol bilayers. In the presence of one VP1 protein, we monitor the formation of small local negative curvature and membrane thinning at the protein binding site as well as reduction of area per lipid. These membrane deformations are also observed under cholesterol-free conditions. However, here, the number of GM1 molecules attached to the VP1 binding pockets increases. The membrane curvature is slightly increased for asymmetric GM1 distribution that mimics conditions in vivo, compared to symmetric GM1 distributions which are often applied in experiments. Slightly smaller inward curvature was observed in atomistic control simulations. Binding of four VP1 proteins leads to an increase of the average intrinsic area per lipid in the protein binding leaflet. Membrane fluctuations appear to be the driving force of VP1 aggregation, as was previously shown for membrane-adhering particles because no VP1 aggregation is observed in the absence of a lipid membrane.

Structure Formation in Langmuir Peptide Films As Revealed from Coarse-Grained Molecular Dynamics Simulations.

Molecular dynamics simulations in conjunction with the Martini coarse-grained model have been used to investigate the (nonequilibrium) behavior of helical 22-residue poly(γ-benzyl-l-glutamate) (PBLG) peptides at the water/vapor interface. Preformed PBLG mono- or bilayers homogeneously covering the water surface laterally collapse in tens of nanoseconds, exposing significant proportions of empty water surface. This behavior was also observed in recent AFM experiments at similar areas per monomer, where a complete coverage had been assumed in earlier work. In the simulations, depending on the area per monomer, either elongated clusters or fibrils form, whose heights (together with the portion of empty water surface) increase over time. Peptides tend to align with respect to the fiber axis or with the major principal axis of the cluster, respectively. The aspect ratio of the cluster observed is 1.7 and, hence, comparable to though somewhat smaller than the aspect ratio of the peptides in α-helical conformation, which is 2.2. The heights of the fibrils is 3 nm after 20 ns and increases to 4.5 nm if the relaxation time is increased by 2 orders of magnitude, in agreement with the experiment. Aggregates with heights of about 3 or 4.5 nm are found to correspond to local bi- or trilayer structures, respectively.

Stable polyglutamine dimers can contain ß-hairpins with interdigitated side chains-but not α-helices, ß-nanotubes, ß-pseudohelices, or steric zippers.

A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the α-helical conformer of polyglutamine is very stable, dimers of α-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, ß-nanotube, and ß-pseudohelix conformers are also too short-lived to initiate aggregation. The ß-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which ß-hairpin-containing conformers act as templates for fibril formation.

Allosteric control of kinesin's motor domain by tubulin: a molecular dynamics study.

Molecular motors such as kinesin are essential for many biological processes. These motors have two motor domains, which bind to tubulin filaments, hydrolyze ATP, and transduce the released chemical energy into directed movements. The general principles of this chemomechanical coupling are now well-established but the underlying molecular mechanisms remain elusive because small conformational changes within large proteins are difficult to detect experimentally. Here, we use atomistic molecular dynamics simulations to monitor such changes within a single motor domain of KIF1A, which belongs to the kinesin-3 motor family. The nucleotide binding pocket of this domain can be empty or occupied by ATP or ADP. For these three nucleotide states, we determine the mobility of the backbone of the protein, both in solution and attached to tubulin. Only one subdomain of the motor domain is found to exhibit a strongly increased mobility upon binding to tubulin: the neck linker that presumably acts as a mechanical transmitter to the other motor domain in dimeric kinesin-3 motors. Furthermore, upon binding to tubulin, the neck linker mobility becomes sensitive to the bound nucleotide and is highly increased after phosphate release, which implies undocking of this linker from the core of the motor domain. These simulation results are consistent with experimental data from EPR spectroscopy, FRET, and cryo-electron microscopy. A detailed analysis of our simulation data also reveals that the undocking of the neck linker in the ADP-kinesin-tubulin state arises from allosteric interactions between the nucleotide and tubulin and that the ß-sheet core undergoes a twist both during phosphate release and ATP binding. The computational approach used here can be applied to other motor domains and mechanoenzymes in order to identify allosteric interactions between the subdomains of these proteins.

Assessing polyglutamine conformation in the nucleating event by molecular dynamics simulations.

Polyglutamine (polyQ) diseases comprise a group of dominantly inherited pathology caused by an expansion of an unstable polyQ stretch which is presumed to form ß-sheets. Similar to other amyloid pathologies, polyQ amyloidogenesis occurs via a nucleated polymerization mechanism, and proceeds through energetically unfavorable nucleus whose existence and structure are difficult to detect. Here, we use atomistic molecular dynamics simulations in explicit solvent to assess the conformation of the polyQ stretch in the nucleus that initiates polyQ fibrillization. Comparison of the kinetic stability of various structures of polyQ peptide with a Q-length in the pathological range (Q40) revealed that steric zipper or nanotube-like structures (ß-nanotube or ß-pseudohelix) are not kinetically stable enough to serve as a template to initiate polyQ fibrillization as opposed to ß-hairpin-based (ß-sheet and ß-sheetstack) or α-helical conformations. The selection of different structures of the polyQ stretch in the aggregation-initiating event may provide an alternative explanation for polyQ aggregate polymorphism.

Antimicrobial selectivity based on zwitterionic lipids and underlying balance of interactions.

An important feature of antimicrobial peptides is their ability to distinguish pro- from eukaryotic membranes. In vitro experiments on the antimicrobial peptide NK-2 indicate that the discrimination between zwitterionic phosphatidylethanolamine lipids exposed by prokaryotes and phosphatidylcholine lipids exposed by eukaryotes plays an important role. The underlying mechanism is not understood. Here we present molecular dynamics simulations in conjunction with a coarse grained model and thermodynamic integration showing that NK-2 binds more strongly to palmitoyloleoylphosphatidylethanolamine (POPE) than to palmitoyloleoylphosphatidylcholine (POPC) bilayers. Finite size effects on the relative free energy have been corrected for with a method that may also be useful in future studies of the affinities of macromolecules for lipid membranes. Our results support the previous hypothesis that the stronger binding to PE compared to PC arises from a better accessibility of the phosphates of the lipids to the cationic peptide in a sense that a similar number of peptide-lipid salt bridges requires to break more favorable electrostatic headgroup-headgroup interactions for PC relative to PE. The transfer of NK-2 from POPC to POPE is found to lead to a decrease in electrostatic peptide-lipid but an increase in lipid-lipid and ion-lipid interactions, correlating with a dehydration of the lipids and the ions but an increased hydration of the peptide. The increase in affinity of NK-2 for POPE compared to POPC hence arises from a complex interplay of competing interactions. This work opens the perspective to study how the affinity of antimicrobial peptides changes with amino acid sequence and lipid composition.

Importance of polar solvation for cross-reactivity of antibody and its variants with steroids.

Understanding the factors determining the binding of ligands to receptors in detail is essential for rational drug design. Here, the free energies of binding of the steroids progesterone (PRG) and 5ß-androstane-3,17-dione (5AD) to the Diels-Alderase antibody 1E9, as well as the Leu(H47)Trp/Arg(H100)Trp 1E9 double mutant (1E9dm) and the corresponding single mutants, have been estimated and decomposed using the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. Also the difference in binding free energies between the PRG-1E9dm complex and the complex of PRG with the antiprogesterone antibody DB3 have been evaluated and decomposed. The steroids bind less strongly to 1E9 than to DB3, but the mutations tend to improve the steroid affinity, in quantitative agreement with experimental data. Although the complexes formed by PRG or 5AD with 1E9dm and by PRG with DB3 have similar affinity, the binding mechanisms are different. Reduced van der Waals interactions as observed for 5AD-1E9dm versus PRG-1E9dm or for PRG-1E9dm versus PRG-DB3 are energetically compensated by an increased solvation of polar groups, partly contrasting previous conclusions based on structural inspection. Our study illustrates that deducing binding mechanisms from structural models alone can be misleading. Therefore, taking into account solvation effects as in MM-PBSA calculations is essential to elucidate molecular recognition.

Model amyloid peptide B18 monomer and dimer studied by replica exchange molecular dynamics simulations.

Peptide misfolding and aggregation are the early steps during the formation of amyloid fibrils. Understanding these processes in detail is crucial for the development of therapeutic strategies against amyloid diseases. Here I present temperature replica exchange molecular dynamics (TREMD) simulations of the model amyloid peptide B18 in the mono- and dimeric states in explicit aqueous solution. Both the monomer and the dimer involve ß-sheets consisting of different residues in different registers with comparable statistical weight. The dimer forms intra- as well as intermolecular ß-sheets. The average ß-sheet content is in agreement with previous estimates from circular dichroism (CD) spectra for monomers and is lower for dimers. The tendency of B18 to form ß-sheets likely contributes to its fibrillogenic property. For both the monomer and the dimer, individual peptides form U-shaped or other partially collapsed conformations. Combined with data from electron microscopy, this suggests that for higher aggregates during fibrillization B18 undergoes a transition from U-shaped to outstretched conformations. The tendency of B18 to form U-shaped conformations, intramolecular ß-sheets, and intermolecular ß-sheets with different register will contribute to the lag phase for fibril formation.

Electrophoresis of neutral oil in water.

Negative electrophoretic mobilities of oil in water are widely interpreted in terms of adsorption of hydroxide leading to negative surface charge. Challenging this traditional view, an increasing body of evidence suggests surface depletion of hydroxide and surface accumulation of hydronium leading to a positive surface charge. We present results from molecular dynamics (MD) simulations showing electrophoretic mobilities of oil in water with the same sign and magnitude as in experiment but in the absence of ions. The underlying mechanism involves interfacial roughness leading to gradients in dielectric permittivity in field direction and, thus, local elevation of the applied electric field. Although all molecules have zero net charge, their partial charges are distributed non-uniformly such that oil exhibits negative and water positive excess charge in regions of high field intensities; this induces a net force on the oil or the water against or in field direction, respectively. Our results indicate that deducing net charges from electrophoretic mobilities as widely done can be misleading. Our findings suggest that pH dependent electrophoretic mobilities in experiment being negative above and positive below pH 2.5 arise from a competition between the negative mobility of the ion-free interface and the positive mobility from adsorbed hydronium ions.

A single bicontinuous cubic phase induced by fusion peptides.

We report a bicontinuous cubic phase forming in the presence of the Influenza HA fusion peptide in coarse grained molecular dynamics simulations. Starting from a random mixture of DOPE, water, and fusion peptides, we observe spontaneous formation of a stable bicontinuous phase. Unlike all previously reported bicontinuous cubic phases the one formed in our simulations is a single phase in the sense that there are no multiple isolated compartments of water or lipid.

Beta-hairpin folding by a model amyloid peptide in solution and at an interface.

The development of specific agents against amyloidoses requires an understanding of the conformational distribution of fibrillogenic peptides at a microscopic level. Here, I present molecular dynamics simulations of the model amyloid peptide LSFD with sequence LSFDNSGAITIG-NH2 in explicit water and at a water/vapor interface for a total time scale of approximately 1.8 micros. An extended structure was used as initial peptide configuration. At approximately 290 K, solvated LSFD was kinetically trapped in diverse misfolded beta-sheet/coil conformations. At 350 K, in contrast, the same type II' beta-hairpin in equilibrium with less ordered but also U-shaped conformations was observed for the core residues DNSGAITI in solution and at the interface in multiple independent simulations. The most stable structural unit of the beta-hairpin was the two residue turn (GA). The core residues exhibited a well-defined folded state in which the beta-hairpin was stabilized by a hydrogen bond between the side chain of Asn-385 and the main chain carbonyl group of Gly-387. My results suggest that beta-sheet conformations indicated from previous Fourier-transform infrared spectroscopy measurements immediately after preparation of the peptide solution may not arise from protofilaments as speculated by others but are a property of LSFD monomers. In addition, combined with previous results from X-ray scattering, my findings suggest that interfacial aggregation of LSFD implies a transition from U-shaped to extended peptide conformations. This work including the first simulations of reversible beta-hairpin folding at an interface is an essential step toward a microscopic understanding of interfacial peptide folding and self-assembly. Knowledge of the main conformation of the peptide core may facilitate the design of possible inhibitors of LSFD aggregation as a test ground for future computational therapeutic strategies against amyloid diseases.

Conformational diversity of the fibrillogenic fusion peptide B18 in different environments from molecular dynamics simulations.

The development of specific agents against amyloidoses requires an understanding of the conformational behavior of fibrillogenic peptides in different environments on the microscopic level. We present extensive molecular dynamics simulations of the fibrillogenic Bindin (103-120) B18 fusion peptide for several different environments: a water-trifluorethanol (TFE) mixture, pure water, aqueous buffer containing 100 mM NaCl, and a buffer-vapor interface. The peptide was studied as an isolated molecule in solution or at an interface. In the simulations, the conformational behavior of the peptide was found to strongly depend on the environment in agreement with experimental data. Overall, large portions of the peptide were unstructured. Preformed alpha-helical conformations were least stable in pure water and most stable in the water-TFE mixture and the buffer-vapor interface. In all environments, the alpha-helical conformation was most stable in the region around residues 113-116, which are mainly hydrophilic. Extended configurations in water or buffer folded into structures containing beta-sheets in agreement with data from circular dichroism spectroscopy. In buffer, the beta-sheet content was larger than in water and alpha-beta transitions were observed at elevated temperature. Beta-sheets were formed by hydrophobic residues; turns were formed by hydrophilic residues. A few typical beta-sheets that contain different residues are suggested. A B18 molecule in a strand-loop-strand conformation placed in buffer in contact with vapor was spontaneously adsorbed to the buffer-vapor interface with its hydrophobic side pointing toward the vapor phase. The adsorption induced the formation of turns at positions 108-119 and alpha-helical conformations in the region around residues 114-117. Alpha-helices were parallel to the interface plane in agreement with data from IR reflection absorption spectroscopy.