Conformational space search of Neuromedin C using replica exchange molecular dynamics and molecular dynamics

The present study involves the utilization of replica exchange molecular dynamics (REMD) methodology to explore the conformational space of Neuromedin C (NMC) using implicit (REMD^implicit) and explicit (REMD^explicit) water models. Comparison of the structures obtained from these simulations indicate that REMD^explicit trajectory display a greater tendency to induce β‐turns and bent structures as compared to those obtained from the REMD^implicit simulation. Moreover, two additional MD trajectories performed using Langevin (MD^Lang) and Berendsen (MD^Berend) algorithms under generalized born (GB) solvent conditions were also suitably competent to sample similar kinds of conformations, although the extent of beta turns was low compared to those observed in REMD^explicit simulation. Finally, the comparison of results obtained from all the trajectories and those derived from the NMR studies of Ni(II) complex of NMC indicates that the REMD under explicit conditions is more efficient in sampling the conformations, and show good agreement with the experimental results. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of force fields on the conformational and dynamic properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1‐40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER‐ff99sb‐ILDN, AMBER‐ff99sb*‐ILDN, AMBER‐ff99sb‐NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER‐ff99sb‐ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α‐helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER‐ff99sb‐NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER‐ff99sb‐NMR force field, the others tended to under estimate the expected amount of β‐sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER‐ff99sb‐NMR, reproduce a theoretically expected β‐sheet‐turn‐β‐sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C‐terminal hydrophobic cores from residues 17‐21 and 30‐36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different.     

Temperature-mediated switching of protectant-denaturant behavior of trimethylamine-N-oxide and consequences on protein stability from a replica exchange molecular dynamics simulation study

The detailed mechanism of protein folding–unfolding processes with the aid of osmolytes has been a leading topic of discussion over many decades. We have used replica-exchange molecular dynamics simulation to propose the molecular mechanism of interaction of a 20-residue mini-protein with urea and trimethylamine N-oxide (TMAO) that act as denaturing and protecting osmolyte, respectively, in binary osmolyte solutions. Urea is found to exert its action by interacting directly with the protein residues. Temperature tolerance of TMAO’s action is particularly emphasised in this study. At lower range of temperature, TMAO acts as a successful protein protectant. Interestingly, the study discloses the tendency of TMAO molecules to prefer self-association at the protein surface at elevated temperature. A greater number of TMAO molecules in the protein hydration shell at higher temperature is also observed. Dihedral angle principal component analysis and free energy landscape plots sampled all possible conformations adopted by the protein that reveal highly folded behaviour of the protein in pure water and binary TMAO solutions and highly unfolded behaviour in presence of urea.     

Union of Geometric Constraint-Based Simulations with Molecular Dynamics for Protein Structure Prediction

Although proteins are a fundamental unit in biology, the mechanism by which proteins fold into their native state is not well understood. In this work, we explore the assembly of secondary structure units via geometric constraint-based simulations and the effect of refinement of assembled structures using reservoir replica exchange molecular dynamics. Our approach uses two crucial features of these methods: i), geometric simulations speed up the search for nativelike topologies as there are no energy barriers to overcome; and ii), molecular dynamics identifies the low free energy structures and further refines these structures toward the actual native conformation. We use eight α-, β-, and α/β-proteins to test our method. The geometric simulations of our test set result in an average RMSD from native of 3.7 Å and this further reduces to 2.7 Å after refinement. We also explore the question of robustness of assembly for inaccurate (shifted and shortened) secondary structure. We find that the RMSD from native is highly dependent on the accuracy of secondary structure input, and even slightly shifting the location of secondary structure along the amino acid sequence can lead to a rapid decrease in RMSD to native due to incorrect packing.     

Aβ Monomers Transiently Sample Oligomer and Fibril-Like Configurations: Ensemble Characterization Using a Combined MD/NMR Approach

Amyloid β (Aβ) peptides are a primary component of fibrils and oligomers implicated in the etiology of Alzheimer's disease (AD). However, the intrinsic flexibility of these peptides has frustrated efforts to investigate the secondary and tertiary structure of Aβ monomers, whose conformational landscapes directly contribute to the kinetics and thermodynamics of Aβ aggregation. In this work, de novo replica exchange molecular dynamics (REMD) simulations on the microseconds-per-replica timescale are used to characterize the structural ensembles of Aβ42, Aβ40, and M35-oxidized Aβ42, three physiologically relevant isoforms with substantially different aggregation properties. J-coupling data calculated from the REMD trajectories were compared to corresponding NMR-derived values acquired through two different pulse sequences, revealing that all simulations converge on the order of hundreds of nanoseconds-per-replica toward ensembles that yield good agreement with experiment. Though all three Aβ species adopt highly heterogeneous ensembles, these are considerably more structured compared to simulations on shorter timescales. Prominent in the C-terminus are antiparallel β-hairpins between L17–A21, A30–L36, and V39–I41, similar to oligomer and fibril intrapeptide models that expose these hydrophobic side chains to solvent and may serve as hotspots for self-association. Compared to reduced Aβ42, the absence of a second β-hairpin in Aβ40 and the sampling of alternate β topologies by M35-oxidized Aβ42 may explain the reduced aggregation rates of these forms. A persistent V24–K28 bend motif, observed in all three species, is stabilized by buried backbone to side-chain hydrogen bonds with D23 and a cross-region salt bridge between E22 and K28, highlighting the role of the familial AD-linked E22 and D23 residues in Aβ monomer folding. These characterizations help illustrate the conformational landscapes of Aβ monomers at atomic resolution and provide insight into the early stages of Aβ aggregation pathways.     

Amyloid beta-protein monomer folding: free-energy surfaces reveal alloform-specific differences

Alloform-specific differences in structural dynamics between amyloid β-protein (Aβ) 40 and Aβ42 appear to underlie the pathogenesis of Alzheimer's disease. To elucidate these differences, we performed microsecond timescale replica-exchange molecular dynamics simulations to sample the conformational space of the Aβ monomer and constructed its free-energy surface. We find that neither peptide monomer is unstructured, but rather that each may be described as a unique statistical coil in which five relatively independent folding units exist, comprising residues 1-5, 10-13, 17-22, 28-37, and 39-42, which are connected by four turn structures. The free-energy surfaces of both peptides are characterized by two large basins, comprising conformers with either substantial α-helix or β-sheet content. Conformational transitions within and between these basins are rapid. The two additional hydrophobic residues at the Aβ42 C-terminus, Ile41 and Ala42, significantly increase contacts within the C-terminus, and between the C-terminus and the central hydrophobic cluster (Leu17-Ala21). As a result, the β-structure of Aβ42 is more stable than that of Aβ40, and the conformational equilibrium in Aβ42 shifts towards β-structure. These results suggest that drugs stabilizing α-helical Aβ conformers (or destabilizing the β-sheet state) would block formation of neurotoxic oligomers. The atomic-resolution conformer structures determined in our simulations may serve as useful targets for this purpose. The conformers also provide starting points for simulations of Aβ oligomerization—a process postulated to be the key pathogenetic event in Alzheimer's disease.     

Structural properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

Replica exchange molecular dynamics simulations (300 ns) were used to study the dimerization of amyloid β(1‐40) (Aβ(1‐40)) polypeptide. Configurational entropy calculations revealed that at physiological temperature (310 K, 37°C) dynamic dimers are formed by randomly docked monomers. Free energy of binding of the two chains to each other was ?93.56 ± 6.341 kJ mol^?1. Prevalence of random coil conformations was found for both chains with the exceptions of increased β‐sheet content from residues 16‐21 and 29‐32 of chain A and residues 15‐21 and 30‐33 of chain B with β‐turn/β‐bend conformations in both chains from residues 1‐16, 21‐29 of chain A, 1‐16, and 21‐29 of chain B. There is a mixed β‐turn/β‐sheet region from residues 33‐38 of both chains. Analysis of intra‐ and interchain residue distances shows that, although the individual chains are highly flexible, the dimer system stays in a loosely packed antiparallel β‐sheet configuration with contacts between residues 17‐21 of chain A with residues 17‐21 and 31‐36 of chain B as well as residues 31‐36 of chain A with residues 17‐21 and 31‐36 of chain B. Based on dihedral principal component analysis, the antiparallel β‐sheet‐loop‐β‐sheet conformational motif is favored for many low energy sampled conformations. Our results show that Aβ(1‐40) can form dynamic dimers in aqueous solution that have significant conformational flexibility and are stabilized by collapse of the central and C‐terminal hydrophobic cores with the expected β‐sheet‐loop‐β‐sheet conformational motif. Proteins 2017; 85:1024–1045. © 2017 Wiley Periodicals, Inc.     

Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Na_v1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Na_v1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Na_v1.4. Here, we determine the NMR structure of a selectively ¹³C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By ¹³C NMR, we obtain NMR structural constraints as ¹³C chemical shifts and the ¹H-²H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the ¹H-²H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel Na_vRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Na_v1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.     

Antigenic Characteristics of Rhinovirus Chimeras Designed in silico for En5hanced Presentation of HIV-1 gp41 Epitopes

The development of an effective AIDS vaccine remains the most promising long-term strategy to combat human immunodeficiency virus (HIV)/AIDS. Here, we report favorable antigenic characteristics of vaccine candidates isolated from a combinatorial library of human rhinoviruses displaying the ELDKWA epitope of the gp41 glycoprotein of HIV-1. The design principles of this library emerged from the application of molecular modeling calculations in conjunction with our knowledge of previously obtained ELDKWA-displaying chimeras, including knowledge of a chimera with one of the best 2F5-binding characteristics obtained to date. The molecular modeling calculations identified the energetic and structural factors affecting the ability of the epitope to assume conformations capable of fitting into the complementarity determining region of the ELDKWA-binding, broadly neutralizing human mAb 2F5. Individual viruses were isolated from the library following competitive immunoselection and were tested using ELISA and fluorescence quenching experiments. Dissociation constants obtained using both techniques revealed that some of the newly isolated chimeras bind 2F5 with greater affinity than previously identified chimeric rhinoviruses. Molecular dynamics simulations of two of these same chimeras confirmed that their HIV inserts were partially preorganized for binding, which is largely responsible for their corresponding gains in binding affinity. The study illustrates the utility of combining structure-based experiments with computational modeling approaches for improving the odds of selecting vaccine component designs with preferred antigenic characteristics. The results obtained also confirm the flexibility of HRV as a presentation vehicle for HIV epitopes and the potential of this platform for the development of vaccine components against AIDS.     

In Silico Vaccine Design Based on Molecular Simulations of Rhinovirus Chimeras Presenting HIV-1 gp41 Epitopes

A cluster of promising epitopes for the development of human immunodeficiency virus (HIV) vaccines is located in the membrane-proximal external region (MPER) of the gp41 subunit of the HIV envelope spike structure. The crystal structure of the peptide corresponding to the so-called ELDKWA epitope (HIV-1 HxB2 gp41 residues 662-668), in complex with the corresponding broadly neutralizing human monoclonal antibody 2F5, provides a target for structure-based vaccine design strategies aimed at finding macromolecular carriers that are able to present this MPER-derived epitope with optimal antigenic activity. To this end, a series of replica exchange molecular dynamics computer simulations was conducted to characterize the distributions of conformations of ELDKWA-based epitopes inserted into a rhinovirus carrier and to identify those with the highest fraction of conformations that are able to bind 2F5. The length, hydrophobic character, and precise site of insertion were found to be critical for achieving structural similarity to the target crystal structure. A construct with a high degree of complementarity to the corresponding determinant region of 2F5 was obtained. This construct was employed to build a high-resolution structural model of the complex between the 2F5 antibody and the chimeric human rhinovirus type 14:HIV-1 ELDKWA virus particle. Additional simulations, which were conducted to study the conformational propensities of the ELDKWA region in solution, confirm the hypothesis that the ELDKWA region of gp41 is highly flexible and capable of assuming helical conformations (as in the postfusion helical bundle structure) and β-turn conformations (as in the complex with the 2F5 antibody). These results also suggest that the ELDKWA epitope can be involved in intramolecular—and likely intermolecular—hydrophobic interactions. This tendency offers an explanation for the observation that mutations decreasing the hydrophobic character of the MPER in many cases result in conformational changes that increase the affinity of this region for the 2F5 antibody.