Modeling the structure of Pyrococcus furiosus rubredoxin by homology to other X-ray structures.

The three-dimensional structure of rubredoxin from the hyperthermophilic archaebacterium, Pyrococcus furiosus, has been modeled from the X-ray crystal structures of three homologous proteins from Clostridium pasteurianum, Desulfovibrio gigas, and Desulfovibrio vulgaris. All three homology models are similar. When comparing the positions of all heavy atoms and essential hydrogen atoms to the recently solved crystal structure (Day, M. W., et al., 1992, Protein Sci. 1, 1494-1507) of the same protein, the homology model differ from the X-ray structure by 2.09 A root mean square (RMS). The X-ray and the zinc-substituted NMR structures (Blake, P. R., et al., 1992b, Protein Sci. 1, 1508-1521) show a similar level of difference (2.05 A RMS). On average, the homology models are closer to the X-ray structure than to the NMR structures (2.09 vs. 2.42 A RMS).     

Refinement of comparative models of protein structure by using multicanonical molecular dynamics simulations

Comparative modelling is a powerful method that easily predicts a considerably accurate structure of a protein by using a template structure having a similar amino-acid sequence to the target protein. However, in the region where the amino-acid sequence is different between the target and the template, the predicted structure remains unreliable. In such a case, the model has to be refined. In the present study, we explored the possibility of a molecular dynamics-based method, using the human SAP Src Homology 2 (SH2) domain as the modelling target. The multicanonical method was used to alleviate the multiple-minima problem and the generalised Born/surface area model was used to reduce the computational cost. In addition, position restraints were imposed on the atoms in the reliable regions to avoid unnecessary conformational sampling. We analyzed the conformational distribution of the ligand-recognition loop of the domain and found that the most populated conformational clusters in the ensemble of the model agreed well with one of the two major clusters in the ensemble of the reference simulation starting from the crystal structure. This demonstrates that the current refinement method can significantly improve the accuracy of an unreliable region in a comparative model.     

Exploring single-domain antibody thermostability by molecular dynamics simulation

Abstract

Single-domain antibodies also known as nanobodies are recombinant antigen-binding domains that correspond to the heavy-chain variable region of camelid antibodies. Previous experimental studies showed that the nanobodies have stable and active structures at high temperatures. In this study, the thermal stability and dynamics of nanobodies have been studied by employing molecular dynamics simulation at different temperatures. Variations in root mean square deviation, native contacts, and solvent-accessible surface area of the nanobodies during the simulation were calculated to analyze the effect of different temperatures on the overall conformation of the nanobody. Then, the thermostability mechanism of this protein was studied through calculation of dynamic cross-correlation matrix, principal component analyses, native contact analyses, and root mean square fluctuation. Our results manifest that the side chain conformation of some residues in the complementarity-determining region 3 (CDR3) and also the interaction between α-helix region of CDR3 and framework2 play a critical role to stabilize the protein at a high temperature.

Communicated by Ramaswamy H. Sarma     

Understanding the effect of tert-butanol on Candida antarctica lipase B using molecular dynamics simulations

The use of organic solvents as reaction media for enzymatic reactions has many advantages. Several organic solvents have been proposed as reaction media, especially for transesterifications using Candida antarctica lipase B (CalB). Among organic solvents, tert-butanol is associated with an enhanced conversion rate in bio-diesel production. Thus, it is necessary to understand the effect of tert-butanol on CalB to explain the high-catalytic efficiency compared with the reaction in other hydrophilic organic solvents. In this study, the effects of tert-butanol on the structure of CalB were investigated by MD simulations. The overall flexibility was increased in the presence of tert-butanol. The substrate entrance and the binding pocket size of CalB in tert-butanol were maintained as in TIP3P water. The distance between the catalytic residues of CalB in tert-butanol indicated a higher likelihood of forming hydrogen bonds. These structural analyses could be useful for understanding the effect of tert-butanol on lipase transesterification.     

Effect of temperature on welding of metallic nanowires investigated using molecular dynamics simulations

The mechanism of welding of Au–Au, Ag–Ag and Au–Ag nanowires (NWs) with head-to-head contact is studied using molecular dynamics simulations based on the second-moment approximation of the many-body tight-binding potential. The effect of temperature in the range of 300–900 K is investigated. Simulation results show that at the initial welding, an incomplete jointing area forms through the interactions of the van der Waals attractive force, and that the jointing area increases with increasing the extent of contact between the two NWs during the welding process and temperature. Few defects form along the (1 1 1) close-packed plane during the welding process because the acting stress is quite low. Among the three NW pairs, the Au–Au NWs have the best cold-welding quality, whereas the Au–Ag NWs have the worst cold-welding quality due to the welding of different materials. With an increase in temperature, the weld stress and the mechanical strength of the NWs significantly decrease, and the number of disordered structures increases. The welding fails when the temperature exceeds the molten temperature of the NWs.     

Exploration of the chlorpyrifos escape pathway from acylpeptide hydrolases using steered molecular dynamics simulations

Acylpeptide hydrolases (APH) catalyze the removal of an N-acylated amino acid from blocked peptides. APH is significantly more sensitive than acetylcholinesterase, a target of Alzheimer’s disease, to inhibition by organophosphorus (OP) compounds. Thus, OP compounds can be used as a tool to probe the physiological functions of APH. Here, we report the results of a computational study of molecular dynamics simulations of APH bound to the OP compounds and an exploration of the chlorpyrifos escape pathway using steered molecular dynamics (SMD) simulations. In addition, we apply SMD simulations to identify potential escape routes of chlorpyrifos from hydrolase hydrophobic cavities in the APH-inhibitor complex. Two previously proposed APH pathways were reliably identified by CAVER 3.0, with the estimated relative importance of P1 > P2 for its size. We identify the major pathway, P2, using SMD simulations, and Arg526, Glu88, Gly86, and Asn65 are identified as important residues for the ligand leaving via P2. These results may help in the design of APH-targeting drugs with improved efficacy, as well as in understanding APH selectivity of the inhibitor binding in the prolyl oligopeptidase family.     

Ligand unbinding pathways from the vitamin D receptor studied by molecular dynamics simulations

Molecular dynamics simulation techniques have been used to study the unbinding pathways of 1α,25-dihydroxyvitamin D₃ from the ligand-binding pocket of the vitamin D receptor (VDR). The pathways observed in a large number of relatively short (<200 ps) random acceleration molecular dynamics (RAMD) trajectories were found to be in fair agreement, both in terms of pathway locations and deduced relative preferences, compared to targeted molecular dynamics (TMD) and streered molecular dynamics simulations (SMD). However, the high-velocity ligand expulsions of RAMD tend to favor straight expulsion trajectories and the observed relative frequencies of different pathways were biased towards the probability of entering a particular exit channel. Simulations indicated that for VDR the unbinding pathway between the H1–H2 loop and the β-sheet between H5 and H6 is more favorable than the pathway located between the H1–H2 loop and H3. The latter pathway has been suggested to be the most likely unbinding path for thyroid hormone receptors (TRs) and a likely path for retinoic acid receptor. Ligand entry/exit through these two pathways would not require displacement of H12 from its agonistic position. Differences in the packing of the H1, H2, H3 and β-sheet region explain the changed relative preference of the two unbinding pathways in VDR and TRs. Based on the crystal structures of the ligand binding domains of class 2 nuclear receptors, whose members are VDR and TRs, this receptor class can be divided in two groups according to the packing of the H1, H2, H3 and β-sheet region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Using molecular dynamics for the refinement of atomistic models of GPCRs by homology modeling

Despite GPCRs sharing a common seven helix bundle, analysis of the diverse crystallographic structures available reveal specific features that might be relevant for ligand design. Despite the number of crystallographic structures of GPCRs steadily increasing, there are still challenges that hamper the availability of new structures. In the absence of a crystallographic structure, homology modeling remains one of the important techniques for constructing 3D models of proteins. In the present study we investigated the use of molecular dynamics simulations for the refinement of GPCRs models constructed by homology modeling. Specifically, we investigated the relevance of template selection, ligand inclusion as well as the length of the simulation on the quality of the GPCRs models constructed. For this purpose we chose the crystallographic structure of the rat muscarinic M3 receptor as reference and constructed diverse atomistic models by homology modeling, using different templates. Specifically, templates used in the present work include the human muscarinic M2; the more distant human histamine H1 and the even more distant bovine rhodopsin as shown in the GPCRs phylogenetic tree. We also investigated the use or not of a ligand in the refinement process. Hence, we conducted the refinement process of the M3 model using the M2 muscarinic as template with tiotropium or NMS docked in the orthosteric site and compared with the results obtained with a model refined without any ligand bound.     

Investigation of ligand selectivity in CYP3A7 by molecular dynamics simulations

Cytochrome P450 (CYP) 3A7 plays a crucial role in the biotransformation of the metabolized endogenous and exogenous steroids. To compare the metabolic capabilities of CYP3A7–ligands complexes, three endogenous ligands were selected, namely dehydroepiandrosterone (DHEA), estrone, and estradiol. In this study, a three-dimensional model of CYP3A7 was constructed by homology modeling using the crystal structure of CYP3A4 as the template and refined by molecular dynamics simulation (MD). The docking method was adopted, combined with MD simulation and the molecular mechanics generalized born surface area method, to probe the ligand selectivity of CYP3A7. These results demonstrate that DHEA has the highest binding affinity, and the results of the binding free energy were in accordance with the experimental conclusion that estrone is better than estradiol. Moreover, several key residues responsible for substrate specificity were identified on the enzyme. Arg372 may be the most important residue due to the low interaction energies and the existence of hydrogen bond with DHEA throughout simulation. In addition, a cluster of Phe residues provides a hydrophobic environment to stabilize ligands. This study provides insights into the structural features of CYP3A7, which could contribute to further understanding of related protein structures and dynamics.     

Sequence dependence of amyloid fibril formation: insights from molecular dynamics simulations

The clarification of the physico-chemical determinants underlying amyloid deposition is critical for our understanding of misfolding diseases. With this purpose we have performed a systematic all-atom molecular dynamics (MD) study of a series of single point mutants of the de novo designed amyloidogenic peptide STVIIE. Sixteen different 50ns long simulations using explicit solvent have been carried out starting from four different conformations of a polymeric six-stranded beta-sheet. The simulations have provided evidence for the influence of a small number of site-specific hydrophobic interactions on the packing and stabilization of nascent aggregates, as well as the interplay between side-chain interactions and the net charge of the molecule on the strand arrangement of polymeric beta-sheets. This MD analysis has also shed light into the origin of the position dependence on mutation of beta-sheet polymerization that was found experimentally for this model system. Our results suggest that MD can be applied to detect critical positions for beta-sheet aggregation within a given amyloidogenic stretch. Studies similar to the one presented here can guide site-directed mutations or the design of drugs that specifically disrupt the key stabilizing interactions of beta-sheet aggregates.     

Understanding the interactions of different substrates with wild-type and mutant acylaminoacyl peptidase using molecular dynamics simulations

Acylaminoacylpeptidase (AAP) belongs to peptidase protein family, which can degrade amyloid β-peptide forms in the brains of patients, and hence leads to Alzheimer’s disease. And so, AAP is considered to be a novel target in the design of drugs against Alzheimer’s disease. In this investigation, six molecular dynamics simulations were used to find that the interaction between the wild-type and R526V AAP with two different substrates (p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide). Our results were as follows: firstly, Ac-Leu-p-nitroanilide bound to R526V AAP to form a more disordered loop (residues 552–562) in the α/β-hydrolase fold like of AAP, which caused an open and inactive AAP domain form, secondly, binding p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide to AAP can decrease the flexibility of residues 225–250, 260–270, and 425–450, in which the ordered secondary structures may contain the suitable geometrical structure and so it is useful to serine attack. Our theoretical results showed that the binding of the two substrates can induce specific conformational changes responsible for the diverse AAP catalytic specificity. These theoretical substrate-induced structural diversities can help explain the abilities of AAPs to recognize and hydrolyze extremely different substrates.     

Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.     

Membrane partitioning of peptide aggregates: coarse-grained molecular dynamics simulations

Abstract

Coarse-grained molecular dynamics (CGMD) simulation technique (MARTINI force field) is applied to monitor the aggregation of helical peptides representing the transmembrane sequence and its extension of bone marrow stromal cell antigen 2 (BST-2). One of the peptides is coupled with a protein transducing domain (PTD) of nine arginine residues (R9) at its N-terminal side as well as a peptide, pep11**, which has been shown to bind to human papilloma virus 16 (HPV16) E6 oncoprotein. A short hydrophobic stretch of the transmembrane domain (TMD) of BST-2 aggregates the fastest and inserts into a lipid membrane. An aggregate of R9-pep11** attaches to the membrane via simultaneous contact of many arginine residues. Monomers from the aggregates of the shortest of the hydrophobic TMDs dissolve into the opposing leaflet when the aggregate spans the bilayer. A ‘flipping’ of the individual monomeric peptides is not observed.

Communicated by Ramaswamy H. Sarma     

Nanocontraction flows of short-chain polyethylene via molecular dynamics simulations

Nanocontraction flows of liquid short-chain polyethylene ([CH₂]₅₀) that were uniformly extruded by a constant-speed piston into a surrounding vacuum from a reservoir through an abrupt contraction nozzle were performed by employing molecular dynamics simulations. The extrudate exhibits a similar die swell phenomenon around the exit of the nozzle. In addition, numerous molecular chains are strongly adsorbed on the external surface of the nozzle. At high extrusion speeds, the velocity and temperature profiles in the nozzle show convex and concave parabolic curves, respectively, whereas the profiles are relatively flat at lower speeds. Near the internal boundary of the nozzle, the wall slip is inspected. Significantly, during the flow, the molecular chains undergo structural deformation, including compressed, stretched and shrunk motions. Comparisons with related experimental observations show that the simulated probability distributions of the bending and dihedral angles, and variations of the squared radius of gyration and orientations, are in reasonable agreement.     

Prediction of the stability of coiled coils using molecular dynamics simulations

We have performed 40–80 ns-long molecular dynamics (MD) simulations of the GCN4 leucine zipper and synthetic coiled coils using the GROMOS96 (43a2) and OPLS-AA force fields, with the aim of predicting coiled coil stability. Starting with an initial configuration of two peptides placed in an ideal coiled coil configuration, we find that changing the amino acid sequence modestly or decreasing peptide length can lead to a decrease in the final α-helicity of coiled coils, although for peptides as long or longer than 16 residues, the values of helicity do not decrease to the low values seen in the experimental results of Lumb et al. (Biochemistry. 1994, 33, 7361–7367) or of Su et al. (Biochemistry. 1994, 33, 15501–15510), presumably because the simulations are not long enough. We find, however, that helicity correlates positively with the number of close hydrophobic interactions between the two peptides, showing that stable coiled coils in the simulations are tightly packed. The minimum interhelical distances are 0.50–0.66 nm for charged groups, indicating that favorable charge interactions are also important for the stability of the coiled coil.     

Brownian dynamics simulations of molecular recognition in an antibody-antigen system.

The crystal structure for an antibody-antigen system, that of the anti-hen egg lysozyme monoclonal antibody HyHEL-5 complexed to lysozyme, is used as the starting point for computer simulations of diffusional encounters between the two proteins. The investigation consists of two parts: first, the linearized Poisson-Boltzmann equation is solved to determine the long-range electrostatic forces between antibody and antigen, and then, the relative motion as influenced by these forces is modeled within Brownian motion theory. The effects of various point mutations on the calculated reaction rate are considered. It is found that charged residues close to the binding site exert the greatest influence in steering the proteins into a configuration favorable for their binding, while more distant mutations are qualitatively described by the Smoluchowski model for the mutual diffusion of two uniformly charged spheres. The antibody residues involved in forming salt links with the lysozyme, Glu-H35 and Glu-H50, appear to be particularly important in electrostatic steering, as neutralization of both of them yields reaction rates that are two to three orders of magnitude below those of wild-type rates. The relative rates obtained from the simulations can be tested through kinetic measurements on mutant protein complexes. Kinetically efficient partners can also be designed and constructed through directed mutagenesis.     

Reconstruction of the src-SH3 protein domain transition state ensemble using multiscale molecular dynamics simulations

We use an integrated computational approach to reconstruct accurately the transition state ensemble (TSE) for folding of the src-SH3 protein domain. We first identify putative TSE conformations from free energy surfaces generated by importance sampling molecular dynamics for a fully atomic, solvated model of the src-SH3 protein domain. These putative TSE conformations are then subjected to a folding analysis using a coarse-grained representation of the protein and rapid discrete molecular dynamics simulations. Those conformations that fold to the native conformation with a probability (P(fold)) of approximately 0.5, constitute the true transition state. Approximately 20% of the putative TSE structures were found to have a P(fold) near 0.5, indicating that, although correct TSE conformations are populated at the free energy barrier, there is a critical need to refine this ensemble. Our simulations indicate that the true TSE conformations are compact, with a well-defined central beta sheet, in good agreement with previous experimental and theoretical studies. A structured central beta sheet was found to be present in a number of pre-TSE conformations, however, indicating that this element, although required in the transition state, does not define it uniquely. An additional tight cluster of contacts between highly conserved residues belonging to the diverging turn and second beta-sheet of the protein emerged as being critical elements of the folding nucleus. A number of commonly used order parameters to identify the transition state for folding were investigated, with the number of native Cbeta contacts displaying the most satisfactory correlation with P(fold) values.