Molecular dynamics simulations of bovine rhodopsin: influence of protonation states and different membrane-mimicking environments

G-protein coupled receptors (GPCRs) are a protein family of outstanding pharmaceutical interest. GPCR homology models, based on the crystal structure of bovine rhodopsin, have been shown to be valuable tools in the drug-design process. The initial model is often refined by molecular dynamics (MD) simulations, a procedure that has been recently discussed controversially. We therefore analyzed MD simulations of bovine rhodopsin in order to identify contacts that could serve as constraints in the simulation of homology models. Additionally, the effect of an N-terminal truncation, the nature of the membrane mimic, the influence of varying protonation states of buried residues and the importance of internal water molecules was analyzed. All simulations were carried out using the program-package GROMACS. While N-terminal truncation negatively influenced the overall protein stability, a stable simulation was possible in both solvent environments. As regards the protonation state of titratable sites, the experimental data could be reproduced by the program UHBD (University of Houston Brownian Dynamics), suggesting its application for studying homology models of GPCRs. A high flexibility was observed for internal water molecules at some sites. Finally, interhelical hydrogen-bonding interactions could be derived, which can now serve as constraints in the simulations of GPCR homology models.     

Molecular dynamics simulations and rigid body (TLS) analysis of aspartate carbamoyltransferase: evidence for an uncoupled R state.

In the R form of ATCase complexed with the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, large temperature factors are reported for the allosteric domains of the regulatory chains. We studied the conformational flexibility of the holoenzyme with molecular dynamics simulations and rigid body (TLS) analysis. The results of the molecular dynamics simulations suggest that, although local atomic fluctuations account for the temperature factors of the catalytic and zinc domains, they do not account for the large temperature factors of the allosteric regions. However, the temperature factors of the allosteric domains can be satisfactorily analyzed using a rigid body model. The simulations and rigid body analysis support the idea that the allosteric regions are mechanically uncoupled from the rest of the enzyme in the PALA structure. Implications of this uncoupling for allosteric regulation are discussed.     

Molecular dynamics simulation of human interleukin-4: comparison with NMR data and effect of pH,counterions and force field on tertiary structure stability

The human protein interleukin-4 (IL-4) has been simulated at two different pH values 2 and 6, with different amounts of counterions present in the aqueous solution, and with two different force-field parameter sets using molecular dynamics simulation with the aim of validation of force field and simulation set-up by comparison to experimental nuclear magnetic resonance data, such as proton–proton nuclear Overhauser effect (NOE) distance bounds, ³ J(HN,HC_α) coupling constants and backbone N–H order parameters. Thirteen simulations varying in the length from 3 to 7 ns are compared.

At pH 6 both force-field parameter sets used do largely reproduce the NOE's and order parameters, the GROMOS 45A3 set slightly better than the GROMOS 53A6 set. ³ J values predicted from the simulation agree less well with experimental values. At pH 2 the protein unfolds, unless counterions are explicitly present in the system, but even then the agreement with experiment is worse than at pH 6. When simulating a highly charged protein, such as IL-4 at pH 2, the inclusion of counterions in the simulation seems mandatory.     

Molecular dynamics simulations of the native and partially folded states of ubiquitin: influence of methanol cosolvent, pH, and temperature on the protein structure and dynamics

A series of explicit-solvent molecular dynamics simulations of the protein ubiquitin are reported, which investigate the effect of environmental factors (presence of methanol cosolvent in the aqueous solution, neutral or low pH value, room or elevated temperature) on the structure, stability, and dynamics of the protein. The simulations are initiated either from the native structure of the protein or from a model of a partially folded state (A-state) that is known to exist at low pH in methanol-water mixtures. The main results of the simulations are: (1) The ubiquitin native structure is remarkably stable at neutral pH in water; (2) the addition of the methanol cosolvent enhances the stability of the secondary structure but weakens tertiary interactions within the protein; (3) this influence of methanol on the protein structure is enhanced at low pH, while the effect of lowering the pH in pure water is limited; and (4) the A-state of ubiquitin can be described as a set of relatively rigid secondary structure elements (a native-like beta-sheet and native-like alpha-helix plus two nonnative alpha-helices) connected by flexible linkers.     

Structure-based analysis of protein dynamics: comparison of theoretical results for hen lysozyme with X-ray diffraction and NMR relaxation data

An analytical approach based on Gaussian network model (GNM) is proposed for predicting the rotational dynamics of proteins. The method, previously shown to successfully reproduce X-ray crystallographic temperature factors for a series of proteins is extended here to predict bond torsional mobilities and reorientation of main chain amide groups probed by 15N-H nuclear magnetic resonance (NMR) relaxation. The dynamics of hen egg-white lysozyme (HEWL) in the folded state is investigated using the proposed approach. Excellent agreement is observed between theoretical results and experimental (X-ray diffraction and NMR relaxation) data. The analysis reveals the important role of coupled rotations, or cross-correlations between dihedral angle librations, in defining the relaxation mechanism on a local scale. The crystal and solution structures exhibit some differences in their local motions, but their global motions are identical. Hinge residues mediating the cooperative movements of the alpha- and beta-domains are identified, which comprise residues in helix C, Glu35 and Ser36 on the loop succeeding helix B, Ile55 and Leu56 at the turn between strands II and III. The central part of the beta-domain long loop and the turn between strands I and II display an enhanced mobility. Finally, kinetically hot residues and key interactions are identified, which point at helix B and beta-strand III as the structural elements underlying the stability of the tertiary structure.     

Molecular identification and functional analysis of Niemann-Pick type C2 protein in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

Niemann-Pick type C2 (NPC2) proteins in arthropods have been extensively differentiated and possibly duplicated according to environmental conditions and are probable to have different functions. The participation of NPC2 proteins in chemical communication in arthropods brings new objectives in environmental-friendly strategies for pest population control. In this study, NPC2 gene in Macrocentrus cingulum (McinNPC2) was newly identified by rapid amplification cDNA ends (RACE) technology. McinNPC2 amino acid sequence alignment with other representative NPC2 annotates to evaluate the highly conserved consensus amino acids, but with odorant binding proteins in M. cingulum show that only one consensus amino acid. Primary six-cysteine structures that are same to odorant binding proteins in M. cingulum were observed in McinNPC2. Phylogenetic analysis of McinNPC2 indicated that the nearest monophyletic group forming one clade with high posterior probability values clusters as Cyphomyrmex costatus (CcosNPC2) whereas the nearest evolutionary relation group as some odorant binding proteins. Moreover, quantitative real-time PCR (qPCR) measurements show that the McinNPC2 gene expression level in various tissues of the female is significantly and ubiquitously higher than in male, whereas the highest expression level in female antennae. We further explore the binding characterization of recombinant McinNPC2 to candidate odor molecules and did the modeling and docking simulations. The results showed ligands binding specificity and docking tests results indicate that β-ionone, an aroma compound commonly found in essential oils, can strongly bind with McinNPC2. In conclusion, we proposed that McinNPC2 may be involved in chemical communication and play roles in perception of plant volatiles.     

Heat shock protein 60 (GroEL) from Porphyromonas gingivalis: Molecular cloning and sequence analysis of its gene and purification of the recombinant protein

Abstract Porphyromonas gingivalis is associated with human periodontal disease. We cloned and sequenced the gene for heat shock protein 60 (GroEL, HSP60) from P. gingivalis FDC381. The identified clone carried a 2.6 kb DNA fragment which contained two open reading frames (ORFs) encoding a 9.6- and a 58.4-kDa protein. The translated amino acid sequence of these ORFs showed a high degree of homology with known sequences for GroES and GroEL from several bacterial species and humans. Escherichia coli carrying this clone expressed a 65-kDa protein which was recognized by anti- Mycobacterium leprae HSP60 monoclonal antibody. We purified the 65-kDa protein by DEAE-sepharose chromatography and hydroxyapatite chromatography. This protein was immunogenic and was recognized by sera from a number of patients with periodontal disease. This immunological reactivity and the existence of molecular mimicry between the P. gingivalis GroEL and other HSP homologs may indicate an important role for this molecule in periodontal lesion.     

Molecular dynamics studies of hexamers of amyloid-beta peptide (16-35) and its mutants: influence of charge states on amyloid formation

To study the early stage of amyloid-beta peptide (Abeta) aggregation, hexamers of the wild-type (WT) Abeta(16-35) and its mutants with amyloid-like conformations have been studied by molecular dynamics simulations in explicit water for a total time of 1.7 micros. We found that the amyloid-like structures in the WT oligomers are destabilized by the solvation of ionic D23/K28 residues, which are buried in the fibrils. This means that the desolvation of D23/K28 residues may contribute to the kinetic barrier of aggregation in the early stage. In the E22Q/D23N, D23N/K28Q, and E22Q/D23N/K28Q mutants, hydration becomes much less significant because the mutated residues have neutral amide side-chains. These amide side-chains can form linear cross-strand hydrogen bond chains, or "polar zippers", if dehydrated. These "polar zippers" increase the stability of the amyloid-like conformation, reducing the barrier for the early-stage oligomerization. This is in accord with experimental observations that both the D23/K28 lactamization and the E22Q/D23N mutation promote aggregation. We also found that the E22Q/D23N mutant prefers an amyloid-like conformation that differs from the one found for WT Abeta. This suggests that different amyloid structures may be formed under different conditions.     

A computational modeling and molecular dynamics study of the Michaelis complex of human protein Z-dependent protease inhibitor (ZPI) and factor Xa (FXa)

Protein Z-dependent protease inhibitor (ZPI) and antithrombin III (AT3) are members of the serpin superfamily of protease inhibitors that inhibit factor Xa (FXa) and other proteases in the coagulation pathway. While experimental structural information is available for the interaction of AT3 with FXa, at present there is no structural data regarding the interaction of ZPI with FXa, and the precise role of this interaction in the blood coagulation pathway is poorly understood. In an effort to gain a structural understanding of this system, we have built a solvent equilibrated three-dimensional structural model of the Michaelis complex of human ZPI/FXa using homology modeling, protein–protein docking and molecular dynamics simulation methods. Preliminary analysis of interactions at the complex interface from our simulations suggests that the interactions of the reactive center loop (RCL) and the exosite surface of ZPI with FXa are similar to those observed from X-ray crystal structure-based simulations of AT3/FXa. However, detailed comparison of our modeled structure of ZPI/FXa with that of AT3/FXa points to differences in interaction specificity at the reactive center and in the stability of the inhibitory complex, due to the presence of a tyrosine residue at the P1 position in ZPI, instead of the P1 arginine residue in AT3. The modeled structure also shows specific structural differences between AT3 and ZPI in the heparin-binding and flexible N-terminal tail regions. Our structural model of ZPI/FXa is also compatible with available experimental information regarding the importance for the inhibitory action of certain basic residues in FXa. Figure Solvent equilibrated models for protein z-dependent protease inhibitor and its initial reactive complex with coagulation factor Xa (show here) are developed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. V.C. and C.J.L. contributed equally to this work. The solvent-equilibrated PDB structure of the ZPI/FXa will be made available upon request. Conflict of interest statement  The authors state that they have no conflict of interest.     

Molecular characterization and phylogenetic analysis of three odorant binding protein gene transcripts in Dendrolimus species (Lepidoptera: Lasiocampidae)

Pine caterpillar moths, Dendrolimus spp. (Lepidoptera: Lasiocampidae), are serious economic pest of pines. Previously, phylogenetic analyses of Dendrolimus using different methods yielded inconsistent results. The chemosensory systems of insects may play fundamental roles in promoting speciation. Odorant‐binding proteins (OBPs) participate in the first step of odor detection. Studying the evolution of OBPs in closely related species may help us to identify their role in speciation. We identified three OBPs – one pheromone‐binding protein and two general odorant‐binding proteins – from male antennae of four Dendrolimus species, D. superans (Butler), D. punctatus (Walker), D. kikuchii Matsumura, and D. houi Lajonquiere, the olfactory recognition systems of which had not been previously investigated. We analyzed their molecular characteristics and compared their sequences to those of OBPs in D. tabulaeformis Tsai et Liu. Ka/Ks ratio analyses among the five Dendrolimus species indicate that PBP1 genes experienced more evolutionary pressure than the GOBPs. Phylogenetic relationships of PBP1 and GOBP1 both indicated that D. houi was the basal species, then branched D. kikuchii, while D. tabulaeformis, D. punctatus, and D. superans evolved more recently. These relationships are consistent with the changes in sex pheromone components of these five species. Dendrolimus tabulaeformis and D. punctatus are closely related sister species. However, the distances among GOBP2 sequences in the five Dendrolimus were very short, and the relationships of D. houi and D. kikuchii could not be resolved. Integrating our results with those of previous studies, we hypothesized that D. kikuchii, D. punctatus and D. superans evolved from the basal ancestor because of sex pheromone mutations and environmental pressure.     

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.     

Coevolving residues of (beta/alpha)(8)-barrel proteins play roles in stabilizing active site architecture and coordinating protein dynamics

Indole-3-glycerol phosphate synthase (IGPS) is a representative of (β/α)₈-barrel proteins—the most common enzyme fold in nature. To better understand how the constituent amino-acids work together to define the structure and to facilitate the function, we investigated the evolutionary and dynamical coupling of IGPS residues by combining statistical coupling analysis (SCA) and molecular dynamics (MD) simulations. The coevolving residues identified by the SCA were found to form a network which encloses the active site completely. The MD simulations showed that these coevolving residues are involved in the correlated and anti-correlated motions. The correlated residues are within van der Waals contact and appear to maintain the active site architecture; the anti-correlated residues are mainly distributed on opposite sides of the catalytic cavity and coordinate the motions likely required for the substrate entry and product release. Our findings might have broad implications for proteins with the highly conserved (βα)₈-barrel in assessing the roles of amino-acids that are moderately conserved and not directly involved in the active site of the (β/α)₈-barrel. The results of this study could also provide useful information for further exploring the specific residue motions for the catalysis and protein design based on the (β/α)₈-barrel scaffold.     

Conformational analysis of the dipeptide taste ligand L-aspartyl-D-2-aminobutyric acid-(S)-α-ethylbenzylamide and its analogues by NMR spectroscopy,computer simulations and X-ray diffraction studies

A dipeptide taste ligand L -aspartyl-D -2-aminobutyric acid-(S)-α-ethylbenzylamide was found to be about 2000 times more potent than sucrose. To investigate the molecular basis of its potent sweet taste, we carried out conformational analysis of this molecule and several related analogues by NMR spectroscopy, computer simulations and X-ray crystallographic studies. The results of the studies support our earlier model that an ‘L’-shape molecular array is essential for eliciting sweet taste. In addition, we have identified an aromatic group located between the stem and the base of the ‘L-shape’, which is responsible for enhancement of sweetness potency. In this study, we also assessed the optimal size of the essential hydrophobic group (X) and the effects of the chirality of the second residue toward taste. ©1997 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular dynamics analysis of the structures of ras-guanine nucleotide exchange protein (SOS) bound to wild-type and oncogenic ras-p21. Identification of effector domains of SOS

The X-ray crystal structure of the ras oncogene-encoded p21 protein bound to SOS, the guanine nucleotide exchange-promoting protein, has been determined. We have undertaken to determine if there are differences between the three-dimensional structures of SOS bound to normal and oncogenic (Val 12-p21) proteins. Using molecular dynamics, we have computed the average structures for both complexes and superimposed them. We find four domains of SOS that differ markedly in structure: 631–641, 676–691, 718–729, and 994–1004. Peptides corresponding to these sequences have been synthesized and found to be powerful modulators of oncogenic p21 in cells as described in an accompanying paper. We find that the SOS segment from 809–815 makes contacts with multiple domains of ras-p21 and can facilitate correlated conformational changes in these domains.     

Identification of a conserved 8 aa insert in the PIP5K protein in the Saccharomycetaceae family of fungi and the molecular dynamics simulations and structural analysis to investigate its potential functional role

Homologs of the phosphatidylinositol‐4‐phosphate‐5‐kinase (PIP5K), which controls a multitude of essential cellular functions, contain a 8 aa insert in a conserved region that is specific for the Saccharomycetaceae family of fungi. Using structures of human PIP4K proteins as templates, structural models were generated of the Saccharomyces cerevisiae and human PIP5K proteins. In the modeled S. cerevisiae PIP5K, the 8 aa insert forms a surface exposed loop, present on the same face of the protein as the activation loop of the kinase domain. Electrostatic potential analysis indicates that the residues from 8 aa conserved loop form a highly positively charged surface patch, which through electrostatic interaction with the anionic portions of phospholipid head groups, is expected to play a role in the membrane interaction of the yeast PIP5K. To unravel this prediction, molecular dynamics (MD) simulations were carried out to examine the binding interaction of PIP5K, either containing or lacking the conserved signature insert, with two different membrane lipid bilayers. The results from MD studies provide insights concerning the mechanistic of interaction of PIP5K with lipid bilayer, and support the contention that the identified 8 aa conserved insert in fungal PIP5K plays an important role in the binding of this protein with membrane surface. Proteins 2017; 85:1454–1467. © 2017 Wiley Periodicals, Inc.     

Molecular dynamics simulation of the effects of single (S221P) and double (S221P and K216E) mutations in the hemagglutinin protein of influenza A H5N1 virus: a study on host receptor specificity

Avian influenza viruses of subtype H5N1 circulating in animals continue to pose threats to human health. The binding preference of the viral surface protein hemagglutinin (HA) to sialosaccharides of receptors is an important area for understanding mutations in the receptor binding site that could be the cause for avian-to-human transmission. In the present work, we studied the effect of two receptor binding site mutations, S221P singly and in combination with another mutation K216E in the HA protein of influenza A H5N1 viruses. Docking of sialic acid ligands corresponding to both avian and human receptors and molecular dynamics simulations of the complexes for wild and mutant strains of H5N1 viruses were carried out. The H5N1 strain possessing the S221P mutation indicated decreased binding to α2,3-linked sialic acids (avian receptor, SAα2,3Gal) when compared to the binding of the wild-type strain that did not possess the HA-221 mutation. The binding to α2,6-linked sialic acids (human receptor, SAα2,6Gal) was found to be comparable, indicating that the mutant strain shows limited dual receptor specificity. On the other hand, the S221P mutation in synergism with the K216E mutation in the binding site, resulted in increased binding affinity for SAα2,6Gal when compared to SAα2,3Gal, indicative of enhanced binding to human receptors. The in-depth study of the molecular interactions in the docked complexes could explain how co-occurring mutations in the HA viral protein can aid in providing fitness advantage to the virus, in the context of host receptor specificity in emerging variants of H5N1 influenza viruses.     

Docking and molecular dynamics simulations of the Fyn‐SH3 domain with free and phospholipid bilayer‐associated 18.5‐kDa myelin basic protein (MBP)—Insights into a noncanonical and fuzzy interaction

The molecular details of the association between the human Fyn‐SH3 domain, and the fragment of 18.5‐kDa myelin basic protein (MBP) spanning residues S38–S107 (denoted as xα2‐peptide, murine sequence numbering), were studied in silico via docking and molecular dynamics over 50‐ns trajectories. The results show that interaction between the two proteins is energetically favorable and heavily dependent on the MBP proline‐rich region (P93‐P98) in both aqueous and membrane environments. In aqueous conditions, the xα2‐peptide/Fyn‐SH3 complex adopts a “sandwich”"‐like structure. In the membrane context, the xα2‐peptide interacts with the Fyn‐SH3 domain via the proline‐rich region and the β‐sheets of Fyn‐SH3, with the latter wrapping around the proline‐rich region in a form of a clip. Moreover, the simulations corroborate prior experimental evidence of the importance of upstream segments beyond the canonical SH3‐ligand. This study thus provides a more‐detailed glimpse into the context‐dependent interaction dynamics and importance of the β‐sheets in Fyn‐SH3 and proline‐rich region of MBP. Proteins 2017; 85:1336–1350. © 2017 Wiley Periodicals, Inc.     

Molecular dynamics and simulation analysis against superoxide dismutase (SOD) target of Micrococcus luteus with secondary metabolites from Bacillus licheniformis recognized by genome mining approach

Micrococcus luteus, also known as M. luteus, is a bacterium that inhabits mucous membranes, human skin, and various environmental sources. It is commonly linked to infections, especially among individuals who have compromised immune systems. M. luteus is capable of synthesizing the enzyme superoxide dismutase (SOD) as a component of its protective response to reactive oxygen species (ROS). This enzyme serves as a promising target for drug development in various diseases. The current study utilized a subtractive genomics approach to identify potential therapeutic targets from M. luteus. Additionally, genome mining was employed to identify and characterize the biosynthetic gene clusters (BGCs) responsible for the production of secondary metabolites in Bacillus licheniformis (B. licheniformis), a bacterium known for its production of therapeutically relevant secondary metabolites. Subtractive genomics resulted in identification of important extracellular protein SOD as a drug target that plays a crucial role in shielding cells from damage caused by ROS. Genome mining resulted in identification of five potential ligands (secondary metabolites) from B. licheniformis such as, Bacillibactin (BAC), Paenibactin (PAE), Fengycin (FEN), Surfactin (SUR) and Lichenysin (LIC). Molecular docking was used to predict and analyze the binding interactions between these five ligands and target protein SOD. The resulting protein–ligand complexes were further analyzed for their motions and interactions of atoms and molecules over 250 ns using molecular dynamics (MD) simulation analysis. The analysis of MD simulations suggests, Bacillibactin as the probable candidate to arrest the activities of SOD. All the five compounds reported in this study were found to act by directly/indirectly interacting with ROS molecules, such as superoxide radicals (O₂–) and hydrogen peroxide (H₂O₂), and transforming them into less reactive species. This antioxidant activity contributes to its protective effects against oxidative stress-induced damage in cells making them likely candidate for various applications, including in the development of antioxidant-based therapies, nutraceuticals, and functional foods.