Novel binding patterns between ganoderic acids and neuraminidase: Insights from docking,molecular dynamics and MM/PBSA studies

Recently, ganoderic acids (GAs) give rise to the attractive candidates of novel neuraminidase (NA) inhibitors. However, there is still no evident conclusion about their binding patterns. To this end, docking, molecular dynamics and MM/PBSA methods were combined to study the binding profiles of GAs with the N1 protein and familiar H274Y and N294S mutations (A/Vietnam/1203/04 stain). It was found that the binding affinities of ganoderic acid DM and Z (ΔG_bind, −16.83 and −10.99 kcal mol⁻¹) are comparable to that of current commercial drug oseltamivir (−23.62 kcal mol⁻¹). Electrostatic interaction is the main driving force, and should be one important factor to evaluate the binding quality and rational design of NA inhibitors. The 150-loop residues Asp151 and Arg152 played an important role in the binding processes. Further analysis revealed that ganoderic acid DM is a potential source of anti-influenza ingredient, with novel binding pattern and advantage over oseltamivir. It had steric hindrance on the 150 cavity of N1 protein, and exerted activities across the H274Y and N294S mutations. This work also pointed out how to effectively design dual-site NA inhibitors and reinforce their affinities. These findings should prove valuable for the in-depth understanding of interactions between NA and GAs, and warrant the experimental aspects to design novel anti-influenza drugs.     

Mechanistic insights into the catalytic reaction of plant allene oxide synthase (pAOS) via QM and QM/MM calculations

QM cluster and QM/MM protein models have been employed to understand aspects of the reaction mechanism of plant allene oxide synthase (pAOS). In this study we have investigated two reaction mechanisms for pAOS. The standard pAOS mechanism was contrasted with an alternative involving an additional active site molecule which has been shown to facilitate proton coupled electron transfer (PCET) in related systems. Firstly, we found that the results from QM/MM protein model are comparable with those from the QM cluster model, presumably due to the large active site used. Furthermore, the results from the QM cluster model show that the Fe^III and Fe^IV pathways for the standard mechanism have similar energetic and structural properties, indicating that the reaction mechanism may well proceed via both pathways. However, while the PCET process is facilitated by an additional active site bound water in other related families, in pAOS it is not, suggesting this type of process is not general to all closely related family members.     

Prediction of the receptor conformation for iGluR2 agonist binding: QM/MM docking to an extensive conformational ensemble generated using normal mode analysis

Highly flexible proteins constitute a significant challenge in molecular docking within the field of drug design. Depending on the efficacy of the bound ligand, the ligand-binding domain (LBD) of the ionotropic glutamate receptor iGluR2 adopts markedly different degrees of domain closure due to large-scale domain movements. With the purpose of predicting the induced domain closure of five known iGluR2 partial to full agonists we performed a validation study in which normal mode analysis (NMA) was employed to generate a 25-membered ensemble of iGluR2 LBD structures with gradually changing domain closures, followed by accurate QM/MM docking to the ensemble. Based on the docking scores we were able to predict the correct optimal degree of closure for each ligand within 1–3° deviation from the experimental structures. We demonstrate that NMA is a useful tool for reliable ensemble generation and that we are able to predict the ligand induced conformational change of the receptor through docking to such an ensemble. The described protocol expands and improves the information that can be obtained from computational docking when dealing with a flexible receptor.     

Prediction of the receptor conformation for iGluR2 agonist binding: QM/MM docking to an extensive conformational ensemble generated using normal mode analysis

Highly flexible proteins constitute a significant challenge in molecular docking within the field of drug design. Depending on the efficacy of the bound ligand, the ligand-binding domain (LBD) of the ionotropic glutamate receptor iGluR2 adopts markedly different degrees of domain closure due to large-scale domain movements. With the purpose of predicting the induced domain closure of five known iGluR2 partial to full agonists we performed a validation study in which normal mode analysis (NMA) was employed to generate a 25-membered ensemble of iGluR2 LBD structures with gradually changing domain closures, followed by accurate QM/MM docking to the ensemble. Based on the docking scores we were able to predict the correct optimal degree of closure for each ligand within 1–3° deviation from the experimental structures. We demonstrate that NMA is a useful tool for reliable ensemble generation and that we are able to predict the ligand induced conformational change of the receptor through docking to such an ensemble. The described protocol expands and improves the information that can be obtained from computational docking when dealing with a flexible receptor.     

Docking,molecular dynamics and free energy studies on aspartoacylase mutations involved in Canavan disease

The disruption of aspartoacylase enzyme’s catalytic activity causes fatal neurodegenerative Canavan disease. By molecular dynamics and docking methods, here we studied two deleterious mutations that have been identified in the Canavan patients’ genotype E285A, F295S, and revealed the possible cause for the enzyme inhibition due to the drastic changes in active site dynamics, loss of interactions among Arg 71, Arg 168 and the substrate and pKa value of critical Glu178 residue. In addition to changes in the enzyme dynamics, free energy calculations show that the binding energy of substrate decreases dramatically up on mutations.     

Binding mechanism of CDK5 with roscovitine derivatives based on molecular dynamics simulations and MM/PBSA methods

Roscovitine derivatives are potent inhibitors of cyclin-dependent kinase 5 (CDK5), but they exhibit different activities, which has not been understood clearly up to now. On the other hand, the task of drug design is difficult because of the fuzzy binding mechanism. In this context, the methods of molecular docking, molecular dynamics (MD) simulation, and binding free energy analysis are applied to investigate and reveal the detailed binding mechanism of four roscovitine derivatives with CDK5. The electrostatic and van der Waals interactions of the four inhibitors with CDK5 are analyzed and discussed. The calculated binding free energies in terms of MM-PBSA method are consistent with experimental ranking of inhibitor effectiveness for the four inhibitors. The hydrogen bonds of the inhibitors with Cys83 and Lys33 can stabilize the inhibitors in binding sites. The van der Waals interactions, especially the pivotal contacts with Ile10 and Leu133 have larger contributions to the binding free energy and play critical roles in distinguishing the variant bioactivity of four inhibitors. In terms of binding mechanism of the four inhibitors with CDK5 and energy contribution of fragments of each inhibitor, two new CDK5 inhibitors are designed and have stronger inhibitory potency.     

QM/MM and SCRF studies of the ionization state of 8-methylpterin substrate bound to dihydrofolate reductase: existence of a low-barrier hydrogen bond

Using combined semiempirical quantum mechanics and molecular mechanics (QM/MM) and ab initio self-consistent reaction field (SCRF) calculations, we determined that a low-barrier hydrogen bond (LBHB) is formed when the mechanism-based substrate 8-methylpterin binds to dihydrofolate reductase (DHFR). The substrate initially was assumed bound either in the ion-pair form corresponding to N3-protonated substrate hydrogen (H) bonded to the unprotonated (carboxylate) of the conserved Glu30 residue in the active site, or in the neutral-pair form corresponding to unprotonated substrate H bonded to the neutral (carboxylic acid) from of Glu30. The free energy of interaction of these H-bonded systems with the protein/solvent surroundings was computed using a coordinate-coupled free energy perturbation (FEP) method implemented within the molecular dynamics (MD) simulation scheme and using a semiempirical (PM3) QM/MM force field. The free energy obtained from the QM/MM force-field simulations corresponds most closely with the corresponding free energy component obtained from HF/6-31G* SCRF calculations using a value of 2 for the dielectric constant (epsilon) for the solvated protein. Calculations were performed at levels ranging from HF/6-31G to MP2/6-31G* to B3LYP/6-31 + G**, with varying dielectric constants. The energy-minimized path for motion of the proton in the H bond along a one-dimensional reaction coordinate was calculated at HF/6-31G, HF/6-31G* (epsilon = 1) and B3LYP/6-31G* (epsilon = 2) levels. These calculations identified a second neutral-pair complex, involving the 2-amino group of substrate, which also interacts with Glu30, which is lower in energy than the ion-pair form. A harmonic vibrational analysis shows that the first vibrational state appears to lie near or above the TS connecting potential energy minima corresponding to the two neutral-pair configurations, thus indicating an LBHB. Consequently, the H-bonded system will have a significant probability of being found in the ion-pair form, in agreement with experimental spectral studies indicating an enzyme-bound cation and suggesting that the LBHB would activate substrate towards hydride-ion transfer from NADPH.     

Multi-scale molecular dynamics study of cholera pentamer binding to a GM1-phospholipid membrane

The AB₅ type toxin produced by the Vibrio cholerae bacterium is the causative agent of the cholera disease. The cholera toxin (CT) has been shown to bind specifically to GM1 glycolipids on the membrane surface. This binding of CT to the membrane is the initial step in its endocytosis and has been postulated to cause significant disruption to the membrane structure. In this work, we have carried out a combination of coarse-grain and atomistic simulations to study the binding of CT to a membrane modelled as an asymmetrical GM1-DPPC bilayer. Simulation results indicate that the toxin binds to the membrane through only three of its five B subunits, in effect resulting in a tilted bound configuration. Additionally, the binding of the CT can increase the area per lipid of GM1 leaflet, which in turn can cause the membrane regions interacting with the bound subunits to experience significant bilayer thinning and lipid tail disorder across both the leaflets.     

Comparative structural studies of psychrophilic and mesophilic protein homologues by molecular dynamics simulation

Comparative molecular dynamics simulations of psychrophilic type III antifreeze protein from the North-Atlantic ocean-pout Macrozoarces americanus and its corresponding mesophilic counterpart, the antifreeze-like domain of human sialic acid synthase, have been performed for 10 ns each at five different temperatures. Analyses of trajectories in terms of secondary structure content, solvent accessibility, intramolecular hydrogen bonds and protein–solvent interactions indicate distinct differences in these two proteins. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their growth temperatures. However at higher temperatures psychrophilic protein shows increased overall flexibility than its mesophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are non-overlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar directions of motion of each protein especially at 298 K, 310 K and 373 K. Overall, the psychrophilic protein leads to increased conformational sampling of the phase space than its mesophilic counterpart.Our study may help in elucidating the molecular basis of thermostability of homologous proteins from two organisms living at different temperature conditions. Such an understanding is required for designing efficient proteins with characteristics for a particular application at desired working temperatures.     

QM/MM modeling of the hydrolysis and transfructosylation reactions of fructosyltransferase from Aspergillus japonicas,an enzyme that produces prebiotic fructooligosaccharide

Fructosyltransferases (FTs) act on sucrose by cleaving the β-(2 → 1) linkage, releasing glucose, and then transferring the fructosyl group to an acceptor molecule. These enzymes are capable of producing prebiotic fructooligosaccharides (FOSs) that are of industrial interest. While several FOS-synthesizing enzymes FTs have been investigated, their catalytic mechanism is not yet fully understood, especially the molecular details of how FOS are enzymatically synthesized from sucrose. Here, we present a comparative quantum mechanics/molecular mechanics (QM/MM) study on the hydrolysis and transfructosylation reactions catalyzed by A. japonicus FT using sucrose as donor and acceptor substrates. It is shown that the hydrolysis and transfructosylation reactions of the enzyme seem to be competitive with similar potential energy profiles. For all studied reaction steps, the fructosyl ring bound in the −1 position was observed to have a ⁴E conformation in the oxocarbonium ion-like transition state. Based on the SCC-DFTB/MM simulations of sucrose complexes of wildtype and D191A mutant FT, Asp191 is shown to be responsible for the productive sugar conformation (at subsite −1) required for catalysis. A key interaction, Asp119⋯nucleophile⋯1–OH (substrate), is proposed to facilitate the formation of fructosyl-enzyme intermediate. This is the first computational study for understanding the FOS synthesis process, and it can be applicable to related FOS-synthesizing enzymes.     

The inhibitory effect of helenalin on telomerase activity is attributed to the alkylation of the CYS445 residue: Evidence from QM/MM simulations

Enhanced telomerase activity is a hallmark in the majority of cancer cells. Thus, understanding the interactions between telomerase and its inhibitors is fundamentally important for the development of novel anticancer drugs without severe side effects. In this study, the covalent binding of helenalin to CYS445 of telomerase (PDB ID: 3DU6) was simulated using combined quantum chemical and molecular mechanical (QM/MM) methods. The results showed that the reaction was a reversible Michael-type addition and a hydrogen bond was formed between helenalin and the side chain of LYS416 of telomerase during the reaction procedure. The LYS416 residue is vital to telomere DNA recognition by interacting with DNA base through hydrogen bonds. The alkylation of CYS445 of telomerase by helenalin may interfere with the telomere DNA recognition at the telomerase active site, thus resulting in inhibition of the enzyme activity.     

Atomic-level molecular modeling of the nonionic surfactant Triton X-100: the OPE9 component in vacuum and water

The commercially available nonionic surfactant Triton X-100 is a mixture of polyoxyethylene tert-octylphenyl ethers (OPE_n) with an average of n = 9.5 oxyethylene (OE) units in the molecules, and the population maximum at n = 9. Thus, the OPE_{n = 9} component was chosen to be studied by atomic level molecular modeling, using second-generation force fields. The 1,000 conformers generated via random sampling of torsional angles around single bonds yielded 11 clusters based on geometrical similarity. Representatives of geometrically distinctly different clusters with significant populations were chosen from a narrow energy range around the most probable energy to be analyzed for interaction with water. The effect of water on the conformation of the OE chain was found to be modest, similar to the situation that had been reported earlier for the anionic surfactant Aerosol-OT (AOT). The number of bound water molecules is strongly dependent on the conformation of the OE chain and is affected by electrostatic as well as steric effects. Unlike the case of AOT, for which the length of the hydrophobic tail was found to govern the size of reverse micelles in CCl₄, the size of reverse micelles of OPE_{n = 9} cannot be predicted from the dimensions of the hydrophilic tail.     

Probing the binding site characteristics of HSA: A combined molecular dynamics and cheminformatics investigation

Human serum albumin is a remarkable protein found in high concentrations in the body. It contains at least seven distinct fatty acid binding sites and two principle sites for drugs. Its primary function is to act as a fatty acid transport system, but it also shows the capacity to bind a diverse range of acidic, neutral and zwitterionic drug molecules. In this paper we investigate the ligand binding selectivity of HSA using cheminformatics analyses and molecular dynamics simulations. We compare and contrast the known ligand binding specificities as obtained from X-ray structural data using PCA, with additional direct analyses of the seven key binding pockets using analyses derived from molecular simulations. We assess both the fatted and defatted states of HSA using 100 ns simulations of the APO and HOLO forms, as well as structures containing one, three and seven myristic acid molecules. We find that differences in fatty acid binding can have a dramatic effect on the flexibility of the protein and also the pocket characteristics. We discuss how the remarkable selectivity of the HSA pockets towards both endogenous fatty acids and exogenous drug molecules is not simply controlled by bulk property effects such as ionization state and lipophilicity.     

Deconstruction of the human connexin 26 hemichannel due to an applied electric field; A molecular dynamics simulation study

Connexins are a 21-member membrane protein family constituting channels evolved in direct communication between adjacent cells by passaging cytoplasmic molecules and ions. Hexametrical assembly of connexin proteins in plasma membrane forms a wide aqueous pore known as connexin hemichannel. These hemichannels mediate cytoplasm and extracellular milieu communication both in many external tissues and in the central nervous system. In this study, a series of molecular dynamics simulations has been performed to investigate the effect of applied static and alternating electric fields on the stability and conformation of human connexin26 hemichannel. The root mean square deviations of C-alpha atoms, the dipole moment distribution, the number of inter-protein hydrogen bonds and the number of water-protein hydrogen bonds were used to assess connexin26 hemichannel stability. In the static field case, our results show that although the lowest field used in this study (0.1 V/nm) does not lead to the hemichannel deconstruction, stronger fields (>0.1 V/nm), however, disrupt the protein structure during the simulations time period. Furthermore, in the alternating case, compared to static field case, field effects on the connexin26 hemichannel conformation are reduced and consequently the protein maintains its native structure for longer times. Specifically, for the highest frequency used in this study (50 GHz), the hemichannel keeps its structure even under the effect of the strongest field (0.4 V/nm). According to our results, the protein secondary structure is preserved in the characteristic times determined for the protein deconstruction. Consequently, we suggest that the protein deconstruction is due to the tertiary and quaternary structure loss.     

Contributions of 3'-overhang to the dissociation of small interfering RNAs from the PAZ domain: molecular dynamics simulation study

RNA interference (RNAi) is a ‘knock-down’ reaction to reduce expression of a specific gene through highly regulated, enzyme-mediated processes. Small interfering RNAs (siRNAs) are RNA molecules that play an effector role in RNAi and can bind the PAZ domains present in Dicer and RISC. We investigated the interaction between the PAZ domain and the siRNA-like duplexes through dissociation molecular dynamics (DMD) simulations. Specifically, we focused on the response of the PAZ domain to various 3′-overhang structures of the siRNA-like duplexes. We found that the siRNA-like duplex with the 3′ UU-overhang made relatively more stable complex with the PAZ domain compared to those with 3′ CC-, AA-, and GG-overhangs. The siRNA-like duplex with UU-overhang was easily dissociated from the PAZ domain once the structural stability of the complex is impaired. Interestingly, the 3′ UU-overhang spent the least time at the periphery region of the binding pocket during the dissociation process, which can be mainly attributable to UU-overhang's smallest number of hydrogen bonds.     

Prediction of the binding mode of biarylpropylsulfonamide allosteric AMPA receptor modulators based on docking, GRID molecular interaction fields and 3D-QSAR analysis

A novel approach of combining flexible molecular docking, GRID molecular interaction fields, analysis of ligand-protein hydrogen bond interactions, conformational energy penalties and 3D-QSAR analysis was used to propose a binding mode in the dimer interface of the iGluR2 receptor for the biarylpropylsulfonamide class of positive allosteric AMPA modulators. Possible binding poses were generated by flexible molecular docking. GRID molecular interaction fields of the binding site, ligand-protein hydrogen bonding interactions and conformational energy penalties were used to select the most likely binding mode. The selected binding poses were subjected to a 3D-QSAR analysis using previously published activity data. The resulting model (2 LVs, R2=0.89, q2=0.61) predicted the activities of the compounds in the test set with a standard deviation on error of prediction of 0.17. The proposed binding mode was validated by interpretation of the PLS-coefficient regions from the 3D-QSAR analysis in terms of interactions between the receptor and the modulators.     

Exploring the molecular basis of the enantioselective binding of penicillin G acylase towards a series of 2-aryloxyalkanoic acids: a docking and molecular dynamics study

In the present paper, molecular modeling studies were undertaken in order to shed light on the molecular basis of the observed enantioselectivity of penicillin G acylase (PGA), a well known enzyme for its industrial applications, towards 16 racemic 2-aryloxyalkanoic acids, which have been reported to affect several biological systems. With this intention docking calculations and MD simulations were performed. Docking results indicated that the (S)-enantiomers establish several electrostatic interactions with SerB1, SerB386 and ArgB263 of PGA. Conversely, the absence of specific polar interactions between the (R)-enantiomers and ArgB263 seems to be the main reason for the different binding affinities observed between the two enantiomers. Results of molecular dynamics simulations demonstrated that polar interactions are responsible for both the ligand affinity and PGA enantiospecificity. Modeling calculations provided possible explanations for the observed enantioselectivity of the enzyme that rationalize available experimental data and could be the basis for future protein engineering efforts.