The maturation of HIV‐1 protease precursor studied by discrete molecular dynamics

The equilibrium properties of a HIV‐1‐protease precursor are studied by means of an efficient molecular dynamics scheme, which allows for the simulation of the folding of the protein monomers and their dimerization into an active form and compare them with those of the mature protein. The results of the model provide, with atomic detail, an overall account of several experimental findings, including the NMR conformation of the mature dimer, the calorimetric properties of the system, the effects of the precursor tail on the dimerization constant, the secondary chemical shifts of the monomer, and the paramagnetic relaxation enhancement data associated with the conformations of the precursor. It is found that although the mature protein can dimerize in a unique, single way, the precursor populates several dimeric conformations in which monomers are always native‐like, but their binding can be non‐native. Proteins 2014; 82:633–639. © 2013 Wiley Periodicals, Inc.     

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     

A statistical approach to the interpretation of molecular dynamics simulations of calmodulin equilibrium dynamics

A sample of 35 independent molecular dynamics (MD) simulations of calmodulin (CaM) equilibrium dynamics was prepared from different but equally plausible initial conditions (20 simulations of the wild-type protein and 15 simulations of the D129N mutant). CaM's radius of gyration and backbone mean-square fluctuations were analyzed for the effect of the D129N mutation, and simulations were compared with experiments. Statistical tests were employed for quantitative comparisons at the desired error level. The computational model predicted statistically significant compaction of CaM relative to the crystal structure, consistent with the results of small-angle X-ray scattering (SAXS) experiments. This effect was not observed in several previously reported studies of (Ca2+)(4)-CaM, which relied on a single MD run. In contrast to radius of gyration, backbone mean-square fluctuations showed a distinctly non-normal and positively skewed distribution for nearly all residues. Furthermore, the D129N mutation affected the backbone dynamics in a complex manner and reduced the mobility of Glu123, Met124, Ile125, Arg126, and Glu127 located in the adjacent alpha-helix G. The implications of these observations for the comparisons of MD simulations with experiments are discussed. The proposed approach may be useful in studies of protein equilibrium dynamics where MD simulations fall short of properly sampling the conformational space, and when the comparison with experiments is affected by the reproducibility of the computational model.     

Probing the "annealing" mechanism of GroEL minichaperone using molecular dynamics simulations

Although the intact chaperonin machinery is needed to rescue natural substrate proteins (SPs) under non-permissive conditions the "minichaperone" alone, containing only the isolated apical domain of GroEL, can assist folding of a certain class of proteins. To understand the annealing function of the minichaperone, we have carried out molecular dynamics simulations in the NPT ensemble totaling 300ns for four systems; namely, the isolated strongly binding peptide (SBP), the minichaperone, and the SBP and a weakly binding peptide (WBP) in complex with the minichaperone. The SBP, which is structureless in isolation, adopts a beta-hairpin conformation in complex with the minichaperone suggesting that favorable non-specific interactions of the SPs confined to helices H and I of the apical domains can induce local secondary structures. Comparison of the dynamical fluctuations of the apo and the liganded forms of the minichaperone shows that the stability (needed for SP capture) involves favorable hydrophobic interactions and hydrogen bond network formation between the SBP and WBP, and helices H and I. The release of the SP, which is required for the annealing action, involves water-mediated interactions of the charged residues at the ends of H and I helices. The simulation results are consistent with a transient binding release (TBR) model for the annealing action of the minichaperone. According to the TBR model, SP annealing occurs in two stages. In the first stage the SP is captured by the apical domain. This is followed by SP release (by thermal fluctuations) that places it in a different region of the energy landscape from which it can partition rapidly to the native state with probability Phi or be trapped in another misfolded state. The process of binding and release can result in enhancement of the native state yield. The TBR model suggests "that any cofactor that can repeatedly bind and release SPs can be effective in assisting protein folding." By comparing the structures of the non-chaperone alpha-casein (which has no sequence similarity with the apical domain) and the minichaperone and the hydrophobicity profiles we show that alpha-casein has a pair of helices that have similar sequence and structural profiles as H and I. Based on this comparison we identify residues that stabilize (destabilize) alpha-casein-protein complexes. This suggests that alpha-casein assists folding by the TBR mechanism.     

Classical molecular interaction potentials: improved setup procedure in molecular dynamics simulations of proteins.

The latest version of the classical molecular interaction potential (CMIP) has the ability to predict the position of crystallographic waters in several proteins with great accuracy. This article analyzes the ability of the CMIP functional to improve the setup procedure of the molecular system in molecular dynamics (MD) simulations of proteins. To this end, the CMIP strategy is used to include both water molecules and counterions in different protein systems. The structural details of the configurations sampled from trajectories obtained using the CMIP setup procedure are compared with those obtained from trajectories derived from a standard equilibration process. The results show that standard MD simulations can lead to artifactual results, which are avoided using the CMIP setup procedure. Because the CMIP is easy to implement at a low computational cost, it can be very useful in obtaining reliable MD trajectories.     

Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study

Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.     

Structural features of cholesteryl ester transfer protein: A molecular dynamics simulation study

Cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl esters (CEs) from atheroprotective high‐density lipoproteins (HDLs) to atherogenic low‐density lipoproteins (LDLs) or very‐low‐density lipoproteins (VLDLs). Inhibition of CETP raises HDL cholesterol (good cholesterol) levels and reduces LDL cholesterol (bad cholesterol) levels, making it a promising drug target for the prevention and treatment of coronary heart disease. Although the crystal structure of CETP has been determined, the molecular mechanism mediating CEs transfer is still unknown, even the structural features of CETP in a physiological environment remain elusive. We performed molecular dynamics simulations to explore the structural features of CETP in an aqueous solution. Results show that the distal portion flexibility of N‐terminal β‐barrel domain is considerably greater in solution than in crystal; conversely, the flexibility of helix X is slightly less. During the simulations the distal end of C‐terminal β‐barrel domain expanded while the hydrophilic surface increasing more than the hydrophobic surface. In addition, a new surface pore was generated in this domain. This surface pore and all cavities in CETP are stable. These results suggest that the formation of a continuous tunnel within CETP by connecting cavities is permitted in solution. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Structural insight into antisense gapmer-RNA oligomer duplexes through molecular dynamics simulations

There is an extensive research carrying out on antisense technology and the molecules entering into clinical trials are increasing rapidly. Phosphorothioate (PS) is a chemical modification in which nonbridged oxygen is replaced with a sulfur, consequently providing resistance against nuclease activity. The 2'-4' conformationally restricted nucleoside has the structural features of both 2'-O-methoxy ethyl RNA (MOE), which shows good toxicity profile, and locked nucleic acid (LNA), which shows good binding affinity towards the target RNA. These modifications have been studied and suggested that they can be a potential therapeutic agents in antisense therapy. Mipomersen (ISIS 301012), which contains the novel nucleoside modification has been used to target to apolipoprotein (Apo B), which reduces LDL cholesterol by 6–41%. In this study, classical molecular dynamics (MD) simulations were performed on six different antisense gapmer/target-RNA oligomer duplexes (LNA-PS-LNA/RNA, RcMOE-PS-RcMOE/RNA, ScMOE-PS-ScMOE/RNA, MOE-PS-MOE/RNA, PS-DNA/RNA and DNA/RNA) to investigate the structural dynamics, stability and solvation properties. The LNA, MOE nucleotides present in respective duplexes are showing the structure of A-form and the PS-DNA nucleotides resemble the structure of B-form helix with respect to some of the helical parameters. Free energy calculations suggest that the oligomer, which contains LNA binds to the RNA strongly than other modifications as shown in experimental results. The MOE modified nucleotide, which although had a lower binding affinity but higher solvent accessible surface area (SASA) compared to the other modifications, may be influencing the toxicity and hence may be used it in Mipomersen, the second antisense molecule which is approved by FDA.

Communicated by Ramaswamy H. Sarma     

CGDB: A database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein.

The structure and dynamics of the fatty acid binding cavity in I-FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I-FABP, the probe occupied cavity volume and surface are 539+/-8 A3 and 428 A2, respectively (1.4 A probe). A total of 31 residues contact the cavity with their side chains. The side-chain cavity surface is partitioned according to the residue type as follows: 36-39% hydrophobic, 21-25% hydrophilic, and 37-43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity-with the exception of Asp74-are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I-FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I-FABP showed global conformational changes of I-FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnanosecond time scale, which are capable of facilitating exchange of I-FABP internal water.     

Characterization of the denaturation of human alpha-lactalbumin in urea by molecular dynamics simulations

Molecular dynamics (MD) simulations were used to characterize the non-cooperative denaturation of the molten globule A-state of human alpha-lactalbumin by urea. A solvent of explicit urea and water molecules was used, corresponding to a urea concentration of approximately 6M. Three simulations were performed at temperatures of 293K, 360K and 400K, with lengths of 2 ns, 8 ns and 8 ns respectively. The results of the simulations were compared with experimental data from NMR studies of human alpha-lactalbumin and related peptides. During the simulations, hydrogen bonds were formed from the protein to both urea and water molecules as intra-protein hydrogen bonds were lost. Urea was shown to compete efficiently with water as both a hydrogen bond donor and acceptor. Radial distribution functions of water and urea around hydrophobic side chain atoms showed a significant increase in urea molecules in the solvation shell as the side chains became exposed during denaturation. A considerable portion of the native-like secondary structure persisted throughout the simulations. However, in the simulations at 360K and 400K, there were substantial changes in the packing of aromatic and other hydrophobic side chains in the protein, and many native contacts were lost. The results suggest that during the non-cooperative denaturation of the molten globule, secondary structure elements are stabilized by non-specific, non-native interactions.     

qNABpredict: Quick,accurate, and taxonomy-aware sequence-based prediction of content of nucleic acid binding amino acids

Protein sequence-based predictors of nucleic acid (NA)-binding include methods that predict NA-binding proteins and NA-binding residues. The residue-level tools produce more details but suffer high computational cost since they must predict every amino acid in the input sequence and rely on multiple sequence alignments. We propose an alternative approach that predicts content (fraction) of the NA-binding residues, offering more information than the protein-level prediction and much shorter runtime than the residue-level tools. Our first-of-its-kind content predictor, qNABpredict, relies on a small, rationally designed and fast-to-compute feature set that represents relevant characteristics extracted from the input sequence and a well-parametrized support vector regression model. We provide two versions of qNABpredict, a taxonomy-agnostic model that can be used for proteins of unknown taxonomic origin and more accurate taxonomy-aware models that are tailored to specific taxonomic kingdoms: archaea, bacteria, eukaryota, and viruses. Empirical tests on a low-similarity test dataset show that qNABpredict is 100 times faster and generates statistically more accurate content predictions when compared to the content extracted from results produced by the residue-level predictors. We also show that qNABpredict's content predictions can be used to improve results generated by the residue-level predictors. We release qNABpredict as a convenient webserver and source code at http://biomine.cs.vcu.edu/servers/qNABpredict/ . This new tool should be particularly useful to predict details of protein–NA interactions for large protein families and proteomes.     

Insights into internal dynamics of 6-phosphogluconolactonase from Trypanosoma brucei studied by nuclear magnetic resonance and molecular dynamics

Nuclear magnetic resonance is used to investigate the backbone dynamics in 6-phosphogluconolactonase from Trypanosoma brucei (Tb6PGL) with (holo-) and without (apo-) 6-phosphogluconic acid as ligand. Relaxation data were analyzed using the model-free approach and reduced spectral density mapping. Comparison of predictions, based on 77 ns molecular dynamics simulations, with the observed relaxation rates gives insight into dynamical properties of the protein and their alteration on ligand binding. Data indicate dynamics changes in the vicinity of the binding site. More interesting is the presence of perturbations located in remote regions of this well-structured globular protein in which no large-amplitude motions are involved. This suggests that delocalized changes in dynamics that occur upon binding could be a general feature of protein-target interactions.     

Root growth dynamics of Nicotiana attenuata seedlings are affected by simulated herbivore attack

Many studies demonstrate resource-based trade-offs between growth and defence on a large timescale. Yet, the short-term dynamics of this growth reaction are still completely unclear, making it difficult to explain growth-defence trade-offs mechanistically. In this study, image-based non-destructive methods were used to quantify root growth reactions happening within hours following simulated herbivore attack. The induction of wound reactions in Nicotiana attenuata in the seedling stage led to transiently decreased root growth rates. Application of the oral secretion of the specialist herbivore Manduca sexta to the leaves led to a transient decrease in root growth that was more pronounced than if a mere mechanical wounding was imposed. Root growth reduction was more pronounced than leaf growth reduction. When fatty acid-amino acid conjugates (FACs) were applied to wounds, root growth reduction occurred in the same intensity as when oral secretion was applied. Timing of the transient growth reduction coincided with endogenous bursts of jasmonate (JA) and ethylene emissions reported in literature. Simulation of a wound response by applying methyl jasmonate (MeJA) led to more prolonged negative effects on root growth. Increased nicotine concentrations, trichome lengths and densities were observed within 72 h in seedlings that were treated with MeJA or that were mechanically wounded. Overall, these reactions indicate that even in a very early developmental stage, the diversion of plant metabolism from primary (growth-sustaining) to secondary (defence-related) metabolism can cause profound alterations of plant growth performance.     

PDZ binding to the BAR domain of PICK1 is elucidated by coarse-grained molecular dynamics

A key regulator of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor traffic, PICK1 is known to interact with over 40 other proteins, including receptors, transporters and ionic channels, and to be active mostly as a homodimer. The current lack of a complete PICK1 structure determined at atomic resolution hinders the elucidation of its functional mechanisms. Here, we identify interactions between the component PDZ and BAR domains of PICK1 by calculating possible binding sites for the PDZ domain of PICK1 (PICK1-PDZ) to the homology-modeled, crescent-shaped dimer of the PICK1-BAR domain using multiplexed replica-exchange molecular dynamics (MREMD) and canonical molecular dynamics simulations with the coarse-grained UNRES force field. The MREMD results show that the preferred binding site for the single PDZ domain is the concave cavity of the BAR dimer. A second possible binding site is near the N-terminus of the BAR domain that is linked directly to the PDZ domain. Subsequent short canonical molecular dynamics simulations used to determine how the PICK1-PDZ domain moves to the preferred binding site on the BAR domain of PICK1 revealed that initial hydrophobic interactions drive the progress of the simulated binding. Thus, the concave face of the BAR dimer accommodates the PDZ domain first by weak hydrophobic interactions and then the PDZ domain slides to the center of the concave face, where more favorable hydrophobic interactions take over.     

Shape-based virtual screening,docking, and molecular dynamics simulations to identify Mtb-ASADH inhibitors

Aspartate β-semialdehyde dehydrogenase (ASADH) is a key enzyme for the biosynthesis of essential amino acids and several important metabolites in microbes. Inhibition of ASADH enzyme is a promising drug target strategy against Mycobacterium tuberculosis (Mtb). In this work, in silico approach was used to identify potent inhibitors of Mtb-ASADH. Aspartyl β-difluorophosphonate (β-AFP), a known lead compound, was used to understand the molecular recognition interactions (using molecular docking and molecular dynamics analysis). This analysis helped in validating the computational protocol and established the participation of Arg99, Glu224, Cys130, Arg249, and His256 amino acids as the key amino acids in stabilizing ligand–enzyme interactions for effective binding, an essential feature is H-bonding interactions with the two arginyl residues at the two ends of the ligand. Best binding conformation of β-AFP was selected as a template for shape-based virtual screening (ZINC and NCI databases) to identify compounds that competitively inhibit the Mtb-ASADH. The top rank hits were further subjected to ADME and toxicity filters. Final filter was based on molecular docking analysis. Each screened molecule carries the characteristics of the highly electronegative groups on both sides separated by an average distance of 6?Å. Finally, the best predicted 20 compounds exhibited minimum three H-bonding interactions with Arg99 and Arg249. These identified hits can be further used for designing the more potent inhibitors against ASADH family. MD simulations were also performed on two selected compounds (NSC4862 and ZINC02534243) for further validation. During the MD simulations, both compounds showed same H-bonding interactions and remained bound to key active residues of Mtb-ASADH.     

How T cell receptors interact with peptide-MHCs: a multiple steered molecular dynamics study

The T-cell receptor (TCR) interaction with antigenic peptides (p) presented by the major histocompatibility complex (MHC) molecule is a key determinant of immune response. In addition, TCR-pMHC interactions offer examples of features more generally pertaining to protein-protein recognition: subtle specificity and cross-reactivity. Despite their importance, molecular details determining the TCR-pMHC binding remain unsolved. However, molecular simulation provides the opportunity to investigate some of these aspects. In this study, we perform extensive equilibrium and steered molecular dynamics simulations to study the unbinding of three TCR-pMHC complexes. As a function of the dissociation reaction coordinate, we are able to obtain converged H-bond counts and energy decompositions at different levels of detail, ranging from the full proteins, to separate residues and water molecules, down to single atoms at the interface. Many observed features do not support a previously proposed two-step model for TCR recognition. Our results also provide keys to interpret experimental point-mutation results. We highlight the role of water both in terms of interface resolvation and of water molecules trapped in the bound complex. Importantly, we illustrate how two TCRs with similar reactivity and structures can have essentially different binding strategies.