Competitive Binding of Viral Nuclear Localization Signal Peptide and Inhibitor Ligands to Importin-α Nuclear Transport Protein.

Venezuelan equine encephalitis virus (VEEV) is a highly virulent pathogen whose nuclear localization signal (NLS) sequence from capsid protein binds to the host importin-α transport protein and blocks nuclear import. We studied the molecular mechanisms by which two small ligands, termed I1 and I2, interfere with the binding of VEEV's NLS peptide to importin-α protein. To this end, we performed all-atom replica exchange molecular dynamics simulations probing the competitive binding of the VEEV coreNLS peptide and I1 or I2 ligand to the importin-α major NLS binding site. As a reference, we used our previous simulations, which examined noncompetitive binding of the coreNLS peptide or the inhibitors to importin-α. We found that both inhibitors completely abrogate the native binding of the coreNLS peptide, forcing it to adopt a manifold of nonnative loosely bound poses within the importin-α major NLS binding site. Both inhibitors primarily destabilize the native coreNLS binding by masking its amino acids rather than competing with it for binding to importin-α. Because I2, in contrast to I1, binds off-site localizing on the edge of the major NLS binding site, it inhibits fewer coreNLS native binding interactions than I1. Structural analysis is supported by computations of the free energies of the coreNLS peptide binding to importin-α with or without competition from the inhibitors. Specifically, both inhibitors reduce the free energy gain from coreNLS binding, with I1 causing significantly larger loss than I2. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for both inhibitors. Consistent with in silico results, the IC50 value for I1 was found to be lower than that for I2. We hypothesize that the inhibitory action of I1 and I2 ligands might be specific to the NLS from VEEV's capsid protein.

Binding and dimerization of PGLa peptides in anionic lipid bilayer studied by replica exchange molecular dynamics.

The 21-residue PGLa peptide is well known for antimicrobial activity attributed to its ability to compromize bacterial membranes. Using all-atom explicit solvent replica exchange molecular dynamics with solute tempering, we studied PGLa binding to a model anionic DMPC/DMPG bilayer at the high peptide:lipid ratio that promotes PGLa dimerization (a two peptides per leaflet system). As a reference we used our previous simulations at the low peptide:lipid ratio (a one peptide per leaflet system). We found that the increase in the peptide:lipid ratio suppresses PGLa helical propensity, tilts the bound peptide toward the bilayer hydrophobic core, and forces it deeper into the bilayer. Surprisingly, at the high peptide:lipid ratio PGLa binding induces weaker bilayer thinning, but deeper water permeation. We explain these effects by the cross-correlations between lipid shells surrounding PGLa that leads to a much diminished efflux of DMPC lipids from the peptide proximity at the high peptide:lipid ratio. Consistent with the experimental data the propensity for PGLa dimerization was found to be weak resulting in coexistence of monomers and dimers with distinctive properties. PGLa dimers assemble via apolar criss-cross interface and become partially expelled from the bilayer residing at the bilayer-water boundary. We rationalize their properties by the dimer tendency to preserve favorable electrostatic interactions between lysine and phosphate lipid groups as well as to avoid electrostatic repulsion between lysines in the low dielectric environment of the bilayer core. PGLa homedimer interface is predicted to be distinct from that involved in PGLa-magainin heterodimers.

Replica Exchange with Hybrid Tempering Efficiently Samples PGLa Peptide Binding to Anionic Bilayer.

We evaluated the utility of a variant of the replica exchange method, a replica exchange with hybrid tempering (REHT), for all-atom explicit water biomolecular simulations and compared it with a more traditional replica exchange with the solute tempering (REST) algorithm. As a test system, we selected a 21-mer antimicrobial peptide PGLa binding to an anionic DMPC/DMPG lipid bilayer. Application of REHT revealed the following binding mechanism. Due to the strong hydrophobic moment, the bound PGLa adopts an extensive helical structure. The binding free energy landscape identifies two major bound states, a metastable surface bound state and a dominant inserted state. In both states, positively charged PGLa amino acids maintain electrostatic interactions with anionic phosphate groups by rotating the PGLa helix around its axis. PGLa binding causes an influx of anionic DMPG and an efflux of zwitterionic DMPC lipids from the peptide proximity. PGLa thins the bilayer and disorders the adjacent fatty acid tails. Deep invasion of water wires into the bilayer hydrophobic core is detected in the inserted peptide state. The analysis of charge density distributions indicated that peptide positive charges are nearly compensated for by lipid negative charges and water dipole ordering, whereas ions play no role in peptide binding. Thus, electrostatic interactions are the key energetic factor in binding cationic PGLa to an anionic DMPC/DMPG bilayer. Comparison of REHT and REST shows that due to exclusion of lipids from tempered partition, REST lags behind REHT in peptide equilibration, particularly, with respect to peptide insertion and helix acquisition. As a result, REST struggles to provide accurate details of PGLa binding, although it still qualitatively maps the bimodal binding mechanism. Importantly, REHT not only equilibrates PGLa in the bilayer faster than REST, but also with less computational effort. We conclude that REHT is a preferable choice for studying interfacial biomolecular systems.

Binding of viral nuclear localization signal peptides to importin-α nuclear transport protein.

Using all-atom replica-exchange molecular dynamics simulations, we mapped the mechanisms of binding of the nuclear localization signal (NLS) sequence from Venezuelan equine encephalitis virus (VEEV) capsid protein to importin-α (impα) transport protein. Our objective was to identify the VEEV NLS sequence fragment that confers native, experimentally resolved binding to impα as well as to study associated binding energetics and conformational ensembles. The two selected VEEV NLS peptide fragments, KKPK and KKPKKE, show strikingly different binding mechanisms. The minNLS peptide KKPK binds non-natively and nonspecifically by adopting five diverse conformational clusters with low similarity to the x-ray structure 3VE6 of NLS-impα complex. Despite the prevalence of non-native interactions, the minNLS peptide still largely binds to the impα major NLS binding site. In contrast, the coreNLS peptide KKPKKE binds specifically and natively, adopting a largely homogeneous binding ensemble with a dominant, highly native-like conformational cluster. The coreNLS peptide retains most of native binding interactions, including π-cation contacts and a tryptophan cage. While KKPK binding is governed by a complex multistate free energy landscape featuring transitions between multiple binding poses, the coreNLS peptide free energy map is simple, exhibiting a single dominant native-like bound basin. We argue that the origin of the coreNLS peptide binding specificity is several electrostatic interactions formed by the two C-terminal amino acids, Lys10 and Glu11, with impα. The coreNLS sequence is then sufficient for native binding, but none of the amino acids flanking minNLS, including Lys10 and Glu11, are strictly necessary for the native pose. Our analyses indicate that the VEEV coreNLS sequence is virtually unique among human and viral proteins interacting with impα making it a potential target for VEEV-specific inhibitors.

Can Free Energy Perturbation Simulations Coupled with Replica-Exchange Molecular Dynamics Study Ligands with Distributed Binding Sites?

Free energy perturbation coupled with replica exchange with solute tempering (FEP/REST) offers a rigorous approach to compute relative free energy changes for ligands. To determine the applicability of FEP/REST for the ligands with distributed binding poses, we considered two alchemical transformations involving three putative inhibitors I0, I1, and I2 of the Venezuelan equine encephalitis virus nuclear localization signal sequence binding to the importin-α (impα) transporter protein. I0 → I1 and I0 → I2 transformations, respectively, increase or decrease the polarity of the parent molecule. Our objective was three-fold─(i) to verify FEP/REST technical performance and convergence, (ii) to estimate changes in binding free energy ΔΔG, and (iii) to determine the utility of FEP/REST simulations for conformational binding analysis. Our results are as follows. First, our FEP/REST implementation properly follows FEP/REST formalism and produces converged ΔΔG estimates. Due to ligand inherent unbinding, the better FEP/REST strategy lies in performing multiple independent trajectories rather than extending their length. Second, I0 → I1 and I0 → I2 transformations result in overall minor changes in inhibitor binding free energy, slightly strengthening the affinity of I1 and weakening that of I2. Electrostatic interactions dominate binding interactions, determining the enthalpic changes. The two transformations cause opposite entropic changes, which ultimately govern binding affinities. Importantly, we confirm the validity of FEP/REST free energy estimates by comparing them with our previous REST simulations, directly probing binding of three ligands to impα. Third, we established that FEP/REST simulations can sample binding ensembles of ligands. Thus, FEP/REST can be applied (i) to study the energetics of the ligand binding without defined poses and showing minor differences in affinities |ΔΔG| ≲ 0.5 kcal/mol and (ii) to collect ligand binding conformational ensembles.

Binding of Venezuelan Equine Encephalitis Virus Inhibitors to Importin-α Receptors Explored with All-Atom Replica Exchange Molecular Dynamics.

Although Venezuelan equine encephalitis virus (VEEV) is a life-threatening pathogen with a capacity for epidemic outbreaks, there are no FDA-approved VEEV antivirals for humans. VEEV cytotoxicity is partially attributed to the formation of a tetrameric complex between the VEEV capsid protein, the nuclear import proteins importin-α and importin-ß, and the nuclear export protein CRM1, which together block trafficking through the nuclear pore complex. Experimental studies have identified small molecules from the CL6662 scaffold as potential inhibitors of the viral nuclear localization signal (NLS) sequence binding to importin-α. However, little is known about the molecular mechanism of CL6662 inhibition. To address this issue, we employed all-atom replica exchange molecular dynamics simulations to probe, in atomistic detail, the binding mechanism of CL6662 ligands to importin-α. Three ligands, including G281-1485 and two congeners with varying hydrophobicities, were considered. We investigated the distribution of ligand binding poses, their locations, and ligand specificities measured by the strength of binding interactions. We found that G281-1485 binds nonspecifically without forming well-defined binding poses throughout the NLS binding site. Binding of the less hydrophobic congener becomes strongly on-target with respect to the NLS binding site but remains nonspecific. However, a more hydrophobic congener is a strongly specific binder and the only ligand out of three to form a well-defined binding pose, while partially overlapping with the NLS binding site. On the basis of free energy estimates, we argue that all three ligands weakly compete with the viral NLS sequence for binding to importin-α in an apparent compromise to preserve host NLS binding. We further show that all-atom replica exchange binding simulations are a viable tool for studying ligands binding nonspecifically without forming well-defined binding poses.

Lysine Acetylation Changes the Mechanism of Aß25-35 Peptide Binding and Dimerization in the DMPC Bilayer.

The impact of Lys28 acetylation on Alzheimer's Aß peptide binding to the lipid bilayer has not been previously studied, either experimentally or computationally. To probe this common post-translational modification, we performed all-atom replica exchange molecular dynamics simulations targeting binding and aggregation of acetylated acAß25-35 peptide within the DMPC bilayer. Using the unmodified Aß25-35 studied previously as a reference, our results can be summarized as follows. First, Lys28 acetylation strengthens the Aß25-35 hydrophobic moment and consequently promotes the helical structure across the peptide extending it into the N-terminus. Second, because Lys28 acetylation disrupts electrostatic contact between Lys28 and lipid phosphate groups, it reduces the binding affinity of acAß25-35 peptides to the DMPC bilayer. Accordingly, although acetylation preserves the bimodal binding featuring a preferred inserted state and a less probable surface bound state, it decreases the stability of the former. Third, acetylation promotes acAß25-35 aggregation and eliminates monomers as thermodynamically viable species. More importantly, acAß25-35 retains as the most thermodynamically stable the inserted dimer with unique head-to-tail helical aggregation interface. However, due to enhanced helix structure, this dimer state becomes less stable and is less likely to propagate into higher order aggregates. Thus, acetylation is predicted to facilitate the formation of low-molecular-weight oligomers. Other post-translational modifications, including phosphorylation and oxidation, reduce helical propensity and have divergent impact on aggregation. Consequently, acetylation, when considered in its totality, has distinct consequences on Aß25-35 binding and aggregation in the lipid bilayer.

De Novo Transmembrane Aggregation of Aß10-40 Peptides in an Anionic Lipid Bilayer.

Using the all-atom model and 10 µs serial replica-exchange molecular dynamics (SREMD), we investigated the binding of Alzheimer's Aß10-40 peptides to the anionic dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) lipid bilayer. Our objective was to probe de novo transmembrane Aß10-40 aggregation and to test the utility of SREMD. Our results are threefold. First, upon binding, Aß10-40 adopts a helical structure in the C-terminus and deeply inserts into the bilayer. Binding is primarily controlled by electrostatic interactions of the peptides with water, ions, and lipids, particularly, anionic DMPG. Second, Aß-bilayer interactions reorganize lipids in the proximity of the bound peptides, causing an influx of DMPG lipids into the Aß binding footprint. Third and most important, computed free energy landscapes reveal that Aß10-40 peptides partition into monomeric and dimeric species. The dimers result from transmembrane aggregation of the peptides and induce a striking lipid density void throughout both leaflets in the bilayer. There are multiple factors stabilizing transmembrane dimers, including van der Waals and steric interactions, electrostatic interactions, and hydrogen bonding, hydration, and entropic gains originating from dimer conformations and lipid disorder. We argue that helix dipole-dipole interactions underestimated in the all-atom force field must be a contributing factor to stabilizing antiparallel transmembrane dimers. We propose that transmembrane aggregates serve as mechanistic links between the populations of extra- and intracellular Aß peptides. From the computational perspective, SREMD is found to be a viable alternative to traditional replica-exchange simulations.

Mechanisms of Binding of Antimicrobial Peptide PGLa to DMPC/DMPG Membrane.

PGLa belongs to a class of antimicrobial peptides showing strong affinity to anionic bacterial membranes. Using all-atom explicit solvent replica exchange molecular dynamics with solute tempering, we studied binding of PGLa to a model anionic dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayer. Due to a strong hydrophobic moment, PGLa upon binding adopts a helical structure and two distinct bound states separated by a significant free energy barrier. In these states, the C-terminus helix is either surface bound or inserted into the bilayer, whereas the N-terminus remains anchored in the bilayer. Analysis of the free energy landscape indicates that the transition between the two states involves a C-terminus helix rotation permitting the peptide to preserve the interactions between cationic Lys amino acids and anionic lipid phosphorus groups. We calculated the free energy of PGLa binding and showed that it is mostly governed by the balance between desolvation of PGLa positive charges and formation of electrostatic PGLa-lipid interactions. PGLa binding induces minor bilayer thinning but causes pronounced lipid redistribution resulting from an influx of DMPG lipids into the binding footprint and efflux of DMPC lipids. Our in silico results rationalize the S-state detected in NMR experiments.

Machine learning-based prediction of drug and ligand binding in BCL-2 variants through molecular dynamics.

Venetoclax is a BH3 (BCL-2 Homology 3) mimetic used to treat leukemia and lymphoma by inhibiting the anti-apoptotic BCL-2 protein thereby promoting apoptosis of cancerous cells. Acquired resistance to Venetoclax via specific variants in BCL-2 is a major problem for the successful treatment of cancer patients. Replica exchange molecular dynamics (REMD) simulations combined with machine learning were used to define the average structure of variants in aqueous solution to predict changes in drug and ligand binding in BCL-2 variants. The variant structures all show shifts in residue positions that occlude the binding groove, and these are the primary contributors to drug resistance. Correspondingly, we established a method that can predict the severity of a variant as measured by the inhibitory constant (Ki) of Venetoclax by measuring the structure deviations to the binding cleft. In addition, we also applied machine learning to the phi and psi angles of the amino acid backbone to the ensemble of conformations that demonstrated a generalizable method for drug resistant predictions of BCL-2 proteins that elucidates changes where detailed understanding of the structure-function relationship is less clear.

Data Mining of Molecular Simulations Suggest Key Amino Acid Residues for Aggregation, Signaling and Drug Action.

Alzheimer's disease, the most common form of dementia, currently has no cure. There are only temporary treatments that reduce symptoms and the progression of the disease. Alzheimer's disease is characterized by the prevalence of plaques of aggregated amyloid ß (Aß) peptide. Recent treatments to prevent plaque formation have provided little to relieve disease symptoms. Although there have been numerous molecular simulation studies on the mechanisms of Aß aggregation, the signaling role has been less studied. In this study, a total of over 38,000 simulated structures, generated from molecular dynamics (MD) simulations, exploring different conformations of the Aß42 mutants and wild-type peptides were used to examine the relationship between Aß torsion angles and disease measures. Unique methods characterized the data set and pinpointed residues that were associated in aggregation and others associated with signaling. Machine learning techniques were applied to characterize the molecular simulation data and classify how much each residue influenced the predicted variant of Alzheimer's Disease. Orange3 data mining software provided the ability to use these techniques to generate tables and rank the data. The test and score module coupled with the confusion matrix module analyzed data with calculations of specificity and sensitivity. These methods evaluating frequency and rank allowed us to analyze and predict important residues associated with different phenotypic measures. This research has the potential to help understand which specific residues of Aß should be targeted for drug development.

Met35 Oxidation Hinders Aß25-35 Peptide Aggregation within the Dimyristoylphosphatidylcholine Bilayer.

Using all-atom explicit solvent replica exchange molecular dynamics simulations, we studied the aggregation of oxidized (ox) Aß25-35 peptides into dimers mediated by the zwitterionic dimyristoylphosphatidylcholine (DMPC) lipid bilayer. By comparing oxAß25-35 aggregation with that observed for reduced and phosphorylated Aß25-35 peptides, we elucidated plausible impact of post-translational modifications on cytotoxicity of Aß peptides involved in Alzheimer's disease. We found that Met35 oxidation reduces helical propensity in oxAß25-35 peptides bound to the lipid bilayer and enhances backbone fluctuations. These factors destabilize the wild-type head-to-tail dimer interface and lower the aggregation propensity. Met35 oxidation diversifies aggregation pathways by adding monomeric species to the bound conformational ensemble. The oxAß25-35 dimer becomes partially expelled from the DMPC bilayer and as a result inflicts limited disruption to the bilayer structure compared to wild-type Aß25-35. Interestingly, the effect of Ser26 phosphorylation is largely opposite, as it preserves the wild-type head-to-tail aggregation interface and strengthens, not weakens, aggregation propensity. The differing effects can be attributed to the sequence locations of these post-translational modifications, since in contrast to Ser26 phosphorylation, Met35 oxidation directly affects the wild-type C-terminal aggregation interface. A comparison with experimental data is provided.

Partitioning of Aß Peptide Fragments into Blood-Brain Barrier Mimetic Bilayer.

We used all-atom replica-exchange umbrella sampling molecular dynamics simulations to investigate the partitioning of the charged tetrapeptide KLVF and its neutral apolar counterpart VVIA into the blood-brain barrier (BBB)-mimetic bilayer. Our findings allowed us to reconstruct the partitioning mechanism for these two Aß peptide fragments. Despite dissimilar sequences, their permeation shares significant common features. Computations of free energies and permeabilities show that partitioning of both peptides is highly unfavorable, ruling out passive transport. The peptides experience multiple rotational transitions within the bilayer and typically cause considerable lipid disorder and bilayer thinning. Near the bilayer midplane, they lose almost entirely their solvation shell and the interactions with the lipid headgroups. The peptides cause complex reorganization within the proximal bilayer region. Upon insertion, they induce striking cholesterol influx reversed by its depletion and the influx of DMPC when the peptides reach the midplane. The differences in partitioning mechanisms are due to the much higher polarity of KLVF peptide, the permeation of which is more unfavorable and which exclusively assumes vertical orientations within the bilayer. In contrast, VVIA positions itself flat between the leaflets, causing minor disorder and even thickening of the BBB-mimetic bilayer. Due to the high density of the cholesterol-rich BBB bilayer, the unfavorable work associated with the peptide insertion provides a significant, but not dominant, contribution to the partition free energy, which is still governed by dehydration and loss of peptide-headgroup interactions. Comparison with experiments indicates that KLVF and VVIA permeation is similar to that of proline tetrapeptide, mannitol, or cimetidine, all of which exhibit no passive transport.

Predicting Genetic Variation Severity Using Machine Learning to Interpret Molecular Simulations.

Distinct missense mutations in a specific gene have been associated with different diseases as well as differing severity of a disease. Current computational methods predict the potential pathogenicity of a missense variant but fail to differentiate between separate disease or severity phenotypes. We have developed a method to overcome this limitation by applying machine learning to features extracted from molecular dynamics simulations, creating a way to predict the effect of novel genetic variants in causing a disease, drug resistance, or another specific trait. As an example, we have applied this novel approach to variants in calmodulin associated with two distinct arrhythmias as well as two different neurodegenerative diseases caused by variants in amyloid-ß peptide. The new method successfully predicts the specific disease caused by a gene variant and ranks its severity with more accuracy than existing methods. We call this method molecular dynamics phenotype prediction model.

Phosphorylation Promotes Aß25-35 Peptide Aggregation within the DMPC Bilayer.

The consequences of phosphorylation of the Aß25-35 peptide at the position Ser26 on its aggregation have not been examined. To investigate them, we performed all-atom replica exchange simulations probing the binding of phosphorylated Aß25-35 (pAß25-35) peptides to the dimyristoyl phosphatidylcholine (DMPC) bilayer and their subsequent aggregation. As a control, we used our previous study of unmodified peptides. We found that phosphorylation moderately reduces the helical propensity in pAß25-35 and its binding affinity to the DMPC bilayer. Phosphorylation preserves the bimodal binding observed for unmodified Aß25-35, which features a preferred inserted state and a less probable surface bound state. Phosphorylation also retains the inserted dimer with a head-to-tail helical aggregation interface as the most thermodynamically stable state. Importantly, this post-translation modification strengthens interpeptide interactions by adding a new aggregation "hot spot" created by cross-bridging phosphorylated Ser26 with water, cationic ions, or Lys28. The cross-bridging constitutes the molecular mechanism behind most structural phosphorylation effects. In addition, phosphorylation eliminates pAß25-35 monomers and diversifies the pool of aggregated species. As a result, it changes the binding and aggregation mechanism by multiplying pathways leading to stable inserted dimers. These findings offer a plausible molecular rationale for experimental observations, including enhanced production of low molecular weight oligomers and cytotoxicity of phosphorylated Aß peptides.

Partitioning of Benzoic Acid into 1,2-Dimyristoyl-sn-glycero-3-phosphocholine and Blood-Brain Barrier Mimetic Bilayers.

Using an all-atom explicit water model and replica exchange umbrella sampling simulations, we investigated the molecular mechanisms of benzoic acid partitioning into two model lipid bilayers. The first was formed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids, whereas the second was composed of an equimolar mixture of DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, palmitoylsphingomyelin, and cholesterol to constitute a blood-brain barrier (BBB) mimetic bilayer. Comparative analysis of benzoic acid partitioning into the two bilayers has revealed qualitative similarities. Partitioning into the DMPC and BBB bilayers is thermodynamically favorable although insertion into the former lowers the free energy of benzoic acid by approximately an additional 1 kcal mol-1. The partitioning energetics for the two bilayers is also largely similar based on the balance of benzoic acid interactions with apolar fatty acid tails, polar lipid headgroups, and water. In both bilayers, benzoic acid retains a considerable number of residual water molecules until reaching the bilayer midplane where it experiences nearly complete dehydration. Upon insertion into the bilayers, benzoic acid undergoes several rotations primarily determined by the interactions with the lipid headgroups. Nonetheless, in addition to the depth of the free energy minimum, the BBB bilayer differs from the DMPC counterpart by a much deeper location of the free energy minimum and the appearance of a high free energy barrier and positioning of benzoic acid near the midplane. Furthermore, DMPC and BBB bilayers exhibit different structural responses to benzoic acid insertion. Taken together, the BBB mimetic bilayer is preferable for an accurate description of benzoic acid partitioning.

Three Popular Force Fields Predict Consensus Mechanism of Amyloid ß Peptide Binding to the Dimyristoylgylcerophosphocholine Bilayer.

Using all-atom explicit water replica-exchange molecular dynamics simulations, we examined the impact of three popular force fields (FF) on the equilibrium binding of Aß10-40 peptide to the dimyristoylgylcerophosphocholine (DMPC) bilayer. The comparison included CHARMM22 protein FF with CHARMM36 lipid FF (C22), CHARMM36m protein FF with CHARMM36 lipid FF (C36), and Amber14SB protein FF with Lipid14 lipid FF (A14). Analysis of Aß10-40 binding to the DMPC bilayer in three FFs revealed a consensus binding mechanism. Its main features include (i) a stable helical structure in the bound peptide, (ii) insertion of the C-terminus and, in part, the central hydrophobic cluster into the bilayer hydrophobic core, (iii) considerable thinning of the DMPC bilayer beneath the bound peptide coupled with significant drop in bilayer density, and (iv) a strong disordering in the DMPC fatty acid tails. Although the three FFs diverge on many details concerning Aß and bilayer conformational ensembles, these discrepancies do not offset the features of the consensus binding mechanism. We compared our findings with other FF evaluations and proposed that an agreement between C22, C36, and A14 is a consequence of a strong ordering effect created by polar-apolar interface in the lipid bilayer. By comparing the consensus Aß binding mechanism with experimental data, we surmise that the three tested FFs largely correctly capture the interactions of Aß peptides with the DMPC lipid bilayer.

Do Cholesterol and Sphingomyelin Change the Mechanism of Aß25-35 Peptide Binding to Zwitterionic Bilayer?

Using replica exchange with solute tempering all-atom molecular dynamics, we studied the equilibrium binding of Aß25-35 peptide to the ternary bilayer composed of an equimolar mixture of dimyristoylphosphatidylcholine (DMPC), N-palmitoylsphingomyelin (PSM), and cholesterol. Binding of the same peptide to the pure DMPC bilayer served as a control. Due to significant C-terminal hydrophobic moment, binding to the ternary and DMPC bilayers promotes helical structure in the peptide. For both bilayers a polarized binding profile is observed, in which the N-terminus anchors to the bilayer surface, whereas the C-terminus alternates between unbound and inserted states. Both ternary and DMPC bilayers feature two Aß25-35 bound states, surface bound, S, and inserted, I, separated by modest free energy barriers. Experimental data are in agreement with our results but indicate that cholesterol impact is Aß fragment dependent. For Aß25-35, we predict that its binding mechanism is independent of the inclusion of PSM and cholesterol into the bilayer.

De novo aggregation of Alzheimer's Aß25-35 peptides in a lipid bilayer.

A potential mechanism of cytotoxicity attributed to Alzheimer's Aß peptides postulates that their aggregation disrupts membrane structure causing uncontrollable permeation of Ca2+ ions. To gain molecular insights into these processes, we have performed all-atom explicit solvent replica exchange with solute tempering molecular dynamics simulations probing aggregation of the naturally occurring Aß fragment Aß25-35 within the DMPC lipid bilayer. To compare the impact produced on the bilayer by Aß25-35 oligomers and monomers, we used as a control our previous simulations, which explored binding of Aß25-35 monomers to the same bilayer. We found that compared to monomeric species aggregation results in much deeper insertion of Aß25-35 peptides into the bilayer hydrophobic core causing more pronounced disruption in its structure. Aß25-35 peptides aggregate by incorporating monomer-like structures with stable C-terminal helix. As a result the Aß25-35 dimer features unusual helix head-to-tail topology supported by a parallel off-registry interface. Such topology affords further growth of an aggregate by recruiting additional peptides. Free energy landscape reveals that inserted dimers represent the dominant equilibrium state augmented by two metastable states associated with surface bound dimers and inserted monomers. Using the free energy landscape we propose the pathway of Aß25-35 binding, aggregation, and insertion into the lipid bilayer.