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 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.

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.

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.

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.

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.

Is the Conformational Ensemble of Alzheimer's Aß10-40 Peptide Force Field Dependent?

By applying REMD simulations we have performed comparative analysis of the conformational ensembles of amino-truncated Aß10-40 peptide produced with five force fields, which combine four protein parameterizations (CHARMM36, CHARMM22*, CHARMM22/cmap, and OPLS-AA) and two water models (standard and modified TIP3P). Aß10-40 conformations were analyzed by computing secondary structure, backbone fluctuations, tertiary interactions, and radius of gyration. We have also calculated Aß10-40 3JHNHα-coupling and RDC constants and compared them with their experimental counterparts obtained for the full-length Aß1-40 peptide. Our study led us to several conclusions. First, all force fields predict that Aß adopts unfolded structure dominated by turn and random coil conformations. Second, specific TIP3P water model does not dramatically affect secondary or tertiary Aß10-40 structure, albeit standard TIP3P model favors slightly more compact states. Third, although the secondary structures observed in CHARMM36 and CHARMM22/cmap simulations are qualitatively similar, their tertiary interactions show little consistency. Fourth, two force fields, OPLS-AA and CHARMM22* have unique features setting them apart from CHARMM36 or CHARMM22/cmap. OPLS-AA reveals moderate ß-structure propensity coupled with extensive, but weak long-range tertiary interactions leading to Aß collapsed conformations. CHARMM22* exhibits moderate helix propensity and generates multiple exceptionally stable long- and short-range interactions. Our investigation suggests that among all force fields CHARMM22* differs the most from CHARMM36. Fifth, the analysis of 3JHNHα-coupling and RDC constants based on CHARMM36 force field with standard TIP3P model led us to an unexpected finding that in silico Aß10-40 and experimental Aß1-40 constants are generally in better agreement than these quantities computed and measured for identical peptides, such as Aß1-40 or Aß1-42. This observation suggests that the differences in the conformational ensembles of Aß10-40 and Aß1-40 are small and the former can be used as proxy of the full-length peptide. Based on this argument, we concluded that CHARMM36 force field with standard TIP3P model produces the most accurate representation of Aß10-40 conformational ensemble.

Binding of Cytotoxic Aß25-35 Peptide to the Dimyristoylphosphatidylcholine Lipid Bilayer.

Aß25-35 is a short, cytotoxic, and naturally occurring fragment of the Alzheimer's Aß peptide. To map the molecular mechanism of Aß25-35 binding to the zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer, we have performed replica exchange with solute tempering molecular dynamics simulations using all-atom explicit membrane and water models. Consequences of sequence truncation on the binding mechanism have been measured by utilizing as a control our previous simulations probing binding of the longer peptide Aß10-40 to the same bilayer. The most intriguing feature of Aß25-35 binding to the DMPC bilayer is a coexistence of two bound states with strikingly different characteristics: a dominant surface-bound state and a less stable inserted state. In the surface-bound state, the peptide samples extended conformations, in which its unbound C-terminal is pointed away from the bilayer. In contrast, in the inserted state, the C-terminal resides deep in the bilayer hydrophobic core. In both states, the N-terminal remains anchored to the bilayer. Free energy landscape analysis reveals that the two states are separated by a moderate barrier, suggesting that Aß25-35 monomer may frequently interconvert between them. The net effect of Aß25-35 binding is a minor impact on the bilayer structure, which contrasts with the considerable bilayer perturbations induced by a longer Aß10-40 peptide penetrating deep into the bilayer core. Therefore, we conclude that the binding mechanisms of Aß25-35 and Aß10-40 peptides are different. Potential implications of our results for Aß25-35 cytotoxicity are discussed. A comparison of experimental data with our findings reveals a good agreement.

The Alzheimer's disease Aß peptide binds to the anionic DMPS lipid bilayer.

We have applied isobaric-isothermal replica exchange molecular dynamics (REMD) and the all-atom explicit water model to study binding of Aß10-40 peptide to the anionic DMPS bilayer. To provide comparison with a zwitterionic bilayer, we used our previous REMD simulations probing binding of the same peptide to the DMPC bilayer. Using two sets of simulations, we comparatively analyzed the equilibrium Aß conformational ensemble, peptide-bilayer interactions, and changes in the bilayer structure induced by Aß binding. Our results are six-fold. (1) Binding to the DMPS bilayer triggers the formation of stable helix in the Aß C-terminal, although the helix-inducing effect caused by DMPS lipids is weaker than that of DMPC. (2) Compared to the DMPC-bound Aß monomer, the anionic bilayer weakens intrapeptide interactions, particularly, formed by charged amino acids. (3) Binding of Aß peptide to the DMPS bilayer is primarily governed by electrostatic interactions between charged amino acids and charged lipid groups. In contrast, these interactions play minor role in Aß binding to the DMPC bilayer. (4) Aß peptide generally resides on the DMPS bilayer surface causing relatively minor bilayer thinning. The opposite scenario applies to Aß binding to the DMPC bilayer. (5) In contrast to DMPC simulations, Aß largely expels anionic lipids from its binding "footprint" forming a ring of charged amino acids mixed with charged lipid groups around the peptide. (6) Aß binding disorders proximal DMPS lipids more strongly than their DMPC counterparts. Our simulations show that Aß monomers fail to perturb anionic or zwitterionic bilayers across both leaflets.

Cholesterol Changes the Mechanisms of Aß Peptide Binding to the DMPC Bilayer.

Using isobaric-isothermal all-atom replica-exchange molecular dynamics (REMD) simulations, we investigated the equilibrium binding of Aß10-40 monomers to the zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer containing cholesterol. Our previous REMD simulations, which studied binding of the same peptide to the cholesterol-free DMPC bilayer, served as a control, against which we measured the impact of cholesterol. Our findings are as follows. First, addition of cholesterol to the DMPC bilayer partially expels the Aß peptide from the hydrophobic core and promotes its binding to bilayer polar headgroups. Using thermodynamic and energetics analyses, we argued that Aß partial expulsion is not related to cholesterol-induced changes in lateral pressure within the bilayer but is caused by binding energetics, which favors Aß binding to the surface of the densely packed cholesterol-rich bilayer. Second, cholesterol has a protective effect on the DMPC bilayer structure against perturbations caused by Aß binding. More specifically, cholesterol reduces bilayer thinning and overall depletion of bilayer density beneath the Aß binding footprint. Third, we found that the Aß peptide contains a single cholesterol binding site, which involves hydrophobic C-terminal amino acids (Ile31-Val36), Phe19, and Phe20 from the central hydrophobic cluster, and cationic Lys28 from the turn region. This binding site accounts for about 76% of all Aß-cholesterol interactions. Because cholesterol binding site in the Aß10-40 peptide does not contain the GXXXG motif featured in cholesterol interactions with the transmembrane domain C99 of the ß-amyloid precursor protein, we argued that the binding mechanisms for Aß and C99 are distinct reflecting their different conformations and positions in the lipid bilayer. Fourth, cholesterol sharply reduces the helical propensity in the bound Aß peptide. As a result, cholesterol largely eliminates the emergence of helical structure observed upon Aß transition from a water environment to the cholesterol-free DMPC bilayer. We explain this effect by the formation of hydrogen bonds between cholesterol and the Aß backbone, which prevent helix formation. Taken together, we expect that our simulations will advance understanding of a molecular-level mechanism behind the role of cholesterol in Alzheimer's disease.

Inclusion of lipopeptides into the DMPC lipid bilayers prevents Aß peptide insertion.

Using an all-atom explicit water model and replica exchange with solute tempering molecular dynamics we have studied the binding of Aß10-40 peptides to the mixed cationic bilayers composed of DMPC lipids and C14-KAAK lipopeptides (LPs). Using as a control our previous replica exchange simulations probing binding of the same peptide to the zwitterionic DMPC bilayer we assessed the impact of lipopeptides on the Aß binding mechanism. We found that binding to the mixed DMPC + LP bilayers does not enhance the Aß helix propensity as much as binding to the pure DMPC bilayers. Tertiary interactions also differ in the peptide bound to the DMPC + LP bilayers due to a reduced helix content, salt bridge disruption, and the formation of new long-range hydrophobic interactions. More importantly, we showed that mixing lipopeptides into the DMPC bilayers prevents Aß10-40 insertion forcing the peptide to reside on the bilayer surface and considerably destabilizes Aß-bilayer interactions leading to the formation of a shallow water layer between the peptide and the bilayer. Furthermore, we demonstrated that Aß10-40 peptides cause minor DMPC + LP bilayer thinning and perturbation beneath their binding footprint. These observations stand in sharp contrast to Aß10-40 binding to the pure DMPC bilayers, which results in deep peptide insertion and significant disruption of the bilayer structure. We argued that lipopeptides expel Aß10-40 peptides from the bilayer due to strong, mostly electrostatic, interfacial interactions introduced by the lipopeptides into the bilayers. We therefore propose that C14-KAAK lipopeptides can be used to engineer lipid bilayers, which withstand binding of Alzheimer's Aß peptides.

Calcium enhances binding of Aß monomer to DMPC lipid bilayer.

Using isobaric-isothermal replica-exchange molecular dynamics and the all-atom explicit-solvent model, we studied the equilibrium binding of Aß monomers to a zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer coincubated with calcium ions. Using our previous replica-exchange molecular dynamics calcium-free simulations as a control, we reached three conclusions. First, calcium ions change the tertiary structure of the bound Aß monomer by destabilizing several long-range intrapeptide interactions, particularly the salt bridge Asp(23)-Lys(28). Second, calcium strengthens Aß peptide binding to the DMPC bilayer by enhancing electrostatic interactions between charged amino acids and lipid polar headgroups. As a result, Aß monomer penetrates deeper into the bilayer, making disorder in proximal lipids and bilayer thinning more pronounced. Third, because calcium ions demonstrate strong affinity to negatively charged amino acids, a considerable influx of calcium into the area proximal to the bound Aß monomer is observed. Consequently, the localizations of negatively charged amino acids and calcium ions in the Aß binding footprint overlap. Based on our data, we propose a mechanism by which calcium ions strengthen Aß-bilayer interactions. This mechanism involves two factors: 1) calcium ions make the DMPC bilayer partially cationic and thus attractive to the anionic Aß peptide; and 2) destabilization of the Asp(23)-Lys(28) salt bridge makes Lys(28) available for interactions with the bilayer. Finally, we conclude that a single Aß monomer does not promote permeation of calcium ions through the zwitterionic bilayer.

Binding of Aß peptide creates lipid density depression in DMPC bilayer.

Using isobaric-isothermal replica exchange molecular dynamics and all-atom explicit water model we study the impact of Aß monomer binding on the equilibrium properties of DMPC bilayer. We found that partial insertion of Aß peptide into the bilayer reduces the density of lipids in the binding "footprint" and indents the bilayer thus creating a lipid density depression. Our simulations also reveal thinning of the bilayer and a decrease in the area per lipid in the proximity of Aß. Although structural analysis of lipid hydrophobic core detects disordering in the orientations of lipid tails, it also shows surprisingly minor structural perturbations in the tail conformations. Finally, partial insertion of Aß monomer does not enhance water permeation through the DMPC bilayer and even causes considerable dehydration of the lipid-water interface. Therefore, we conclude that Aß monomer bound to the DMPC bilayer fails to perturb the bilayer structure in both leaflets. Limited scope of structural perturbations in the DMPC bilayer caused by Aß monomer may constitute the molecular basis of its low cytotoxicity.

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.

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.