CYP 2D6 Binding Affinity Predictions Using Multiple Ligand and Protein Conformations

Because of the large flexibility and malleability of Cytochrome P450 enzymes (CYPs), in silico prediction of CYP binding affinities to drugs and other xenobiotic compounds is a true challenge. In the current work, we use an iterative linear interaction energy (LIE) approach to compute CYP binding affinities from molecular dynamics (MD) simulation. In order to improve sampling of conformational space, we combine results from simulations starting with different relevant protein-ligand geometries. For calculated binding free energies of a set of thiourea compounds binding to the flexible CYP 2D6 isoform, improved correlation with experiment was obtained by combining results ofMDruns starting from distinct protein conformations and ligand-binding orientations. This accuracy was obtained from relatively short MD simulations, which makes our approach computationally attractive for automated calculations of ligand-binding affinities to flexible proteins such as CYPs.     

A Comparative Study to Decipher the Structural and Dynamics Determinants Underlying the Activity and Thermal Stability of GH-11 Xylanases

With the growing need for renewable sources of energy, the interest for enzymes capable of biomass degradation has been increasing. In this paper, we consider two different xylanases from the GH-11 family: the particularly active GH-11 xylanase from Neocallimastix patriciarum, NpXyn11A, and the hyper-thermostable mutant of the environmentally isolated GH-11 xylanase, EvXyn11^TS. Our aim is to identify the molecular determinants underlying the enhanced capacities of these two enzymes to ultimately graft the abilities of one on the other. Molecular dynamics simulations of the respective free-enzymes and enzyme–xylohexaose complexes were carried out at temperatures of 300, 340, and 500 K. An in-depth analysis of these MD simulations showed how differences in dynamics influence the activity and stability of these two enzymes and allowed us to study and understand in greater depth the molecular and structural basis of these two systems. In light of the results presented in this paper, the thumb region and the larger substrate binding cleft of NpXyn11A seem to play a major role on the activity of this enzyme. Its lower thermal stability may instead be caused by the higher flexibility of certain regions located further from the active site. Regions such as the N-ter, the loops located in the fingers region, the palm loop, and the helix loop seem to be less stable than in the hyper-thermostable EvXyn11^TS. By identifying molecular regions that are critical for the stability of these enzymes, this study allowed us to identify promising targets for engineering GH-11 xylanases. Eventually, we identify NpXyn11A as the ideal host for grafting the thermostabilizing traits of EvXyn11^TS.     

Conformational Landscape of Cytochrome P450 Reductase Interactions

Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein–protein docking to investigate the conformational changes necessary for the formation of the CPR–CYP complex. While some structural features of the CPR and the CPR–CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR–CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR–CYP interactions and underlying conformational changes, improving our understanding of this complex machinery relevant for drug metabolism.     

Conformational Ensembles Explored Dynamically from Disordered Peptides Targeting Chemokine Receptor CXCR4

This work reports on the design and the synthesis of two short linear peptides both containing a few amino acids with disorder propensity and an allylic ester group at the C-terminal end. Their structural properties were firstly analyzed by means of experimental techniques in solution such as CD and NMR methods that highlighted peptide flexibility. These results were further confirmed by MD simulations that demonstrated the ability of the peptides to assume conformational ensembles. They revealed a network of transient and dynamic H-bonds and interactions with water molecules. Binding assays with a well-known drug-target, i.e., the CXCR4 receptor, were also carried out in an attempt to verify their biological function and the possibility to use the assays to develop new specific targets for CXCR4. Moreover, our data indicate that these peptides represent useful tools for molecular recognition processes in which a flexible conformation is required in order to obtain an interaction with a specific target.     

Synthesis of poly(N-isopropylacrylamide) copolymer containing anhydride and imide comonomers - A theoretical study on reversal of LCST

The stimuli sensitive copolymer NIPAM-co-MI was prepared by copolymerizing NIPAM (N-isopropylacrylamide) with varying concentrations of maleimide (MI). The copolymer showed the same ratio of the monomeric components as that of the initial monomer feed ratio, with the two components arranged in the chain in a purely random sequence. Interestingly, the lower critical solution temperature (LCST) of NIPAM-co-MI was found to decrease with increase in MI loading in the copolymer. This behavior was in drastic contrast to the LCST behavior of a similar copolymer NIPAM-co-MA of NIPAM and maleic anhydride (MA) where the LCST showed an increase with increase in the MA concentration. A theoretical interpretation of the contrasting LCST behavior of both NIPAM-co-MI and NIPAM-co-MA was obtained by quantum mechanical (QM) modeling on small structural units of the polymers as well as molecular dynamic (MD) simulations at LCST and above the LCST on 50-unit oligomer model of the polymers. The QM models showed that the MI based polymer is more inclined towards bend structure, higher hydration, and higher intramolecular hydrogen bond formation between its monomer units when compared to those of the MA based polymer. The results of the large scale MD simulation was in complete support of the QM results as it showed the formation of a more folded and highly hydrated NIPAM-co-MI than NIPAM-co-MA.     

Atomistic molecular simulations of structure and dynamics of crosslinked epoxy resin

Many excellent thermal and mechanical performances of cured epoxy resin products can be related to their specific network structure. In this work, a typical crosslinked epoxy resin was investigated using detailed molecular dynamics (MD) simulations, in a wide temperature range from 250 K to 600 K. A general constant-NPT MD procedure widely used for linear polymers failed to identify the glass transition temperature (T_g) of this crosslinked polymer. This can be attributed to the bigger difference in the time scales and cooling rates between the experiments and simulations, and specially to the highly crosslinked infinite network feature. However, by adopting experimental densities appropriate for the corresponding temperatures, some important structural and dynamic features both below and above T_g were revealed using constant-NVT MD simulations. The polymer system exhibited more local structural features in case of below T_g than above T_g, as suggested by some typical radial distribution functions and torsion angle distributions. Non-bond energy, not any other energy components in the used COMPASS forcefield, played the most important role in glass transition. An abrupt change occurring in the vicinity of T_g was also observed in the plots of the mean squared displacements (MSDs) of the crosslinks against the temperature, indicating the great importance of crosslinks to glass transition. Rotational dynamics of some bonds in epoxy segments were also investigated, which exhibited great diversity along the chains between crosslinks. The reorientation functions of these bond vectors at higher temperatures can be well fitted by Kohlrausch-Williams-Watts (KWW) function.     

Post-Translational Modifications of Circulating Alpha-1-Antitrypsin Protein

Alpha-1-antitrypsin (AAT), an acute-phase protein encoded by the SERPINA1 gene, is a member of the serine protease inhibitor (SERPIN) superfamily. Its primary function is to protect tissues from enzymes released during inflammation, such as neutrophil elastase and proteinase 3. In addition to its antiprotease activity, AAT interacts with numerous other substances and has various functions, mainly arising from the conformational flexibility of normal variants of AAT. Therefore, AAT has diverse biological functions and plays a role in various pathophysiological processes. This review discusses major molecular forms of AAT, including complex, cleaved, glycosylated, oxidized, and S-nitrosylated forms, in terms of their origin and function.     

Potential Activity of Fevicordin-A from Phaleria macrocarpa (Scheff) Boerl. Seeds as Estrogen Receptor Antagonist Based on Cytotoxicity and Molecular Modelling Studies

Fevicordin-A (FevA) isolated from Phaleria macrocarpa (Scheff) Boerl. seeds was evaluated for its potential anticancer activity by in vitro and in silico approaches. Cytotoxicity studies indicated that FevA was selective against cell lines of human breast adenocarcinoma (MCF-7) with an IC₅₀ value of 6.4 μM. At 11.2 μM, FevA resulted in 76.8% cell death of T-47D human breast cancer cell lines. Critical pharmacophore features amongst human Estrogen Receptor-α (hERα) antagonists were conserved in FevA with regard to a hypothesis that they could make notable contributions to its pharmacological activity. The binding stability as well as the dynamic behavior of FevA towards the hERα receptor in agonist and antagonist binding sites were probed using molecular dynamics (MD) simulation approach. Analysis of MD simulation suggested that the tail of FevA was accountable for the repulsion of the C-terminal of Helix-11 (H11) in both agonist and antagonist receptor forms. The flexibility of loop-534 indicated the ability to disrupt the hydrogen bond zipper network between H3 and H11 in hERα. In addition, MM/GBSA calculation from the molecular dynamic simulations also revealed a stronger binding affinity of FevA in antagonistic action as compared to that of agonistic action. Collectively, both the experimental and computational results indicated that FevA has potential as a candidate for an anticancer agent, which is worth promoting for further preclinical evaluation.     

氧气和一氧化碳在人血红蛋白迁移过程研究

为了揭示CO和O₂竞争性结合人血红蛋白血红素位点的机制及其与人血红蛋白结构转换之间的关系，本文采用全原子分子动力学模拟（MD）结合马尔科夫状态模型（MSMs）研究氧气（O₂）和一氧化碳（CO）分子从水溶液迁移进入人血红蛋白四聚体α链和β链的全过程。分子动力学模拟揭示了O₂和CO结合α链和β链的稳态结合位点和瞬态结合位点、迁移通道以及α链的结构变化。结果显示，分子模拟不仅仅能够再现全部实验中所观察到的离散Xe结合位点和分子扩散通道，而且揭示了实验中无法观测的瞬态结合位点和多重气体迁移途径。上述结果表明人血红蛋白因其结构柔性所形成的瞬态通道对于气体分子迁移过程的重要性。除此之外，利用MSM和过渡路径理论（TPT）构建了人血红蛋白α链结构变化与气体分子迁移之间的关系，阐释了血红蛋白中影响气体迁移的关键结构及其微观机制。     

Scaling properties in the molecular structure of three-dimensional, nanosized phenylene-based dendrimers as studied by atomistic molecular dynamics simulations

Three-dimensional polyphenylene dendrimers (PDs) can be prepared in ways that enable control of their shape. Their structures may be used as scaffolds with a wide variety of functionality, enabling them to be used as functional nanoparticles with a large range of possible applications, ranging from light emitting devices to biological sensors or drug delivery tools. As PDs have been synthesized only recently, their structural and chemico-physical characterization is still in its infancy. Accordingly, in this paper the shape and internal organization of three PD families based on three different cores were probed by accurate, atomistic molecular dynamics simulations (MD). Particular care was taken to ensure complete structural equilibration by implementing an MD simulated annealing protocol prior to evaluation of the molecular structure and dynamics. All dendrimer families were found to be characterized by molecular dimensions in the nano-range, and by a shape-persistent, non-spherical structure, of molecular fractal dimension around 2.5-2.6, and of surface fractal dimension practically constant and almost equal to 2 with increasing generations in all cases. The MD analysis revealed also that, for this type of dendrimers, the starburst limited generation is presumably located in correspondence of the third generation.     

Optimization to Low Temperature Activity in Psychrophilic Enzymes

Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts.     

Composition-Dependent Dielectric Properties of DMF-Water Mixtures by Molecular Dynamics Simulations

In this paper, we study the dielectric properties of water-N,N dimethylformamide (DMF) mixtures over the whole composition range using a molecular dynamics (MD) simulation. The static and microwave frequency-dependent dielectric properties of the mixtures are calculated from MD trajectories of at least 2 ns length and compared to those of available measurements. We find that the short-ranged structural correlation between neighboring water and DMF molecules strongly influences the static dielectric properties of mixtures. In terms of the dynamics, we report time correlation functions for the dipole densities of mixtures and find that their long-time behavior can be reasonably described by biexponential decays, which means the dielectric relaxations of these mixtures are governed by complex multitimescale mechanisms of rotational diffusion. The dipole density relaxation time is a non-monotonic function of composition passing through a maximum around 0.5 mole fraction DMF, in agreement with the measured main dielectric relaxation time of mixtures.     

Impact of Reactive Oxygen and Nitrogen Species Produced by Plasma on Mdm2–p53 Complex

The study of protein–protein interactions is of great interest. Several early studies focused on the murine double minute 2 (Mdm2)–tumor suppressor protein p53 interactions. However, the effect of plasma treatment on Mdm2 and p53 is still absent from the literature. This study investigated the structural changes in Mdm2, p53, and the Mdm2–p53 complex before and after possible plasma oxidation through molecular dynamic (MD) simulations. MD calculation revealed that the oxidized Mdm2 bounded or unbounded showed high flexibility that might increase the availability of tumor suppressor protein p53 in plasma-treated cells. This study provides insight into Mdm2 and p53 for a better understanding of plasma oncology.     

Molecular dynamics simulation study of the effect of PMMA tacticity on free volume morphology in membranes

The effect of the tacticity of poly methyl methacrylate (PMMA) on the morphology of free volume in PMMA membranes was studied by using a molecular dynamics simulation technique. The chain flexibility, chain interaction, end-to-end distance of a chain and the time course of the free volume variation in the various stereo-regular PMMA, were obtained by an MD simulation technique. Simulation results depict that the construction of distinct tacticity has a significant influence on the flexibility of a molecular chain and the morphology of free volume in the syndiotactic (s-) and isotactic (i-) PMMA oligomers. MD calculation of the dynamic microstructure of a PMMA matrix shows that the s-PMMA membrane gives less flexibility in the backbone and a longer end-to-end distance than in the i-PMMA membrane. The less flexibility and longer end-to-end distance in the s-PMMA matrix causes the shapes and sizes of s-PMMA free volume to be longer and larger than those in an i-PMMA membrane. Furthermore, by adopting conformation energy minimization and molecular dynamics simulation techniques, various tacticity models of PMMA were constructed and their effect on the size of accessible free volume and free volume morphology was analyzed. Finally, the adsorbed gas on the surface free volume in s-PMMA was also compared with i-PMMA membrane.     

Parameterization of classical nonpolarizable force field for hydroxide toward the large-scale molecular dynamics simulation of cellulose in pre-cooled alkali/urea aqueous solution

The empirical force fields (FFs) based on molecular dynamics (MD) simulation studying the dissolution mechanism of cellulose in cold alkali solution suffers the lacking of reliable classical FFs for hydroxide. By a simple adjustment, we transferred one available polarizable force field (FF) of hydroxide into a nonpolarizable one and combined it with GORMOS FF. Simulation based on these parameters provided accurate hydration spheres and solution structure of hydroxide that is comparable to the polarizable one, providing an opportunity for the large-scale MD simulation of the long cellulose chain in alkali/urea system for the study of dissolution and regeneration as well as mercerization process.     

Predicting the Structure and Dynamics of Membrane Protein GerAB from Bacillus subtilis

Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore’s inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine activation of GerA requires all three subunits, but which binds L-alanine is unknown. To date, how GRs trigger germination is unknown, in particular due to lack of detailed structural information about B subunits. Using homology modelling with molecular dynamics (MD) simulations, we present structural predictions for the integral membrane protein GerAB. These predictions indicate that GerAB is an α-helical transmembrane protein containing a water channel. The MD simulations with free L-alanine show that alanine binds transiently to specific sites on GerAB. These results provide a starting point for unraveling the mechanism of L-alanine mediated signaling by GerAB, which may facilitate early events in spore germination.     

Molecular dynamics simulation studies on Ca2+ -induced conformational changes of annexin I

Cryo-electron microscopy (EM) and X-ray studies proposed differentmechanisms for annexin-induced membrane aggregation. In thiswork, molecular dynamics (MD) simulation technique was utilizedto gain an insight into the calcium-induced conformational changeson annexin I and their implication in membrane aggregation mechanism.MD simulations were performed on the Ca²⁺-free annexin I withthe N-terminal domain buried inside the core (System 1), theCa²⁺-bound annexin I without N-terminal domain (System 2) andthe Ca²⁺-bound annexin I with the N-terminal domain exposed(System 3). Our results indicated that calcium binding increasesthe flexibility of annexin I core domain residues includingthe calcium coordinating residues. As a result, annexin I wasactivated to interact with the negatively charged membrane.The exposed N-terminal domain was very flexible and graduallylost the secondary structure during MD simulation, suggestingthat the N-terminal may adopt a favorable conformation to binda second membrane and also explaining the failure of attemptsto crystallize the full-length annexin I in the presence ofcalcium ions. The measured dimensions of the averaged simulationstructure of the Ca²⁺-bound annexin I with the N-terminal exposed(System 3) support the proposed membrane aggregation mechanismbased on X-ray studies.     

Effect of cross-linker length on the thermal and volumetric properties of cross-linked epoxy networks: A molecular simulation study

Volumetric and thermal properties of cross-linked epoxy systems consisting of diglycidyl ether of bisphenol A (DGEBA) and poly(oxypropylene) (POP) diamines of four different lengths ranging from 3 to 68 units were investigated by molecular dynamics (MD) simulations. The cross-linked structures were built by using the simulated annealing polymerization approach. The density, coefficients of volume thermal expansion and glass transition temperature (T_g) of each of the four cross-linked epoxy systems were obtained from their volume–temperature behavior. The density obtained in the simulations agreed well with the experimental value, whereas the coefficients of volume thermal expansion were at least 30% lower than their corresponding experimental results. The predicted T_g values were higher than the experimental values due to the considerably faster cooling rates that are used in the simulations. It was observed that an increase in the chain length of the cross-linker POP-diamines led to a larger difference between the predicted and experimental values of T_g. Three different approaches were used to estimate the expected shift in the experimental T_g to higher values had these measurements been made at cooling rates comparable to those used in MD simulations. It is shown that, in general, the T_g values obtained in MD simulations are consistent with such shifted T_g values that account for the difference in the cooling rates, although no one particular shift approach worked well for all four epoxy systems studied.     

Computational Insights into the Deleterious Impacts of Missense Variants on N-Acetyl-d-glucosamine Kinase Structure and Function

An enzyme of the mammalian amino-sugar metabolism pathway, N-acetylglucosamine kinase (NAGK), that synthesizes N-acetylglucosamine (GlcNAc)-6-phosphate, is reported to promote dynein functions during mitosis, axonal and dendritic growth, cell migration, and selective autophagy, which all are unrelated to its enzyme activity. As non-enzymatic structural functions can be altered by genetic variation, we made an effort in this study aimed at deciphering the pathological effect of nonsynonymous single-nucleotide polymorphisms (nsSNPs) in NAGK gene. An integrated computational approach, including molecular dynamics (MD) simulation and protein–protein docking simulation, was used to identify the damaging nsSNPs and their detailed structural and functional consequences. The analysis revealed the four most damaging variants (G11R, G32R, G120E, and A156D), which are highly conserved and functional, positioned in both small (G11R and G32R) and large (G120E and A156D) domains of NAGK. G11R is located in the ATP binding region, while variants present in the large domain (G120E and A156D) were found to induce substantial alterations in the structural organizations of both domains, including the ATP and substrate binding sites. Furthermore, all variants were found to reduce binding energy between NAGK and dynein subunit DYNLRB1, as revealed by protein–protein docking and MM-GBSA binding energy calculation supporting their deleteriousness on non-canonical function. We hope these findings will direct future studies to gain more insight into the role of these variants in the loss of NAGK function and their role in neurodevelopmental disorders.     

Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold

A subtilisin excreted by the Antarctic Bacillus TA39 has been purified to homogeneity and characterised. Two independent genes subt1 and subt2 are present but only subt1 is expressed significantly in the culture medium. The enzyme displays the usual characteristics of cold enzymes i.e. a high catalytic efficiency at low and moderate temperatures and an increased thermosensitivity originating from a 3D structure probably more flexible than its mesophilic counterpart. This is corroborated by the analysis of the computerized structure which shows a significant decrease in the number and strength of intramolecular weak bonds such as salt bridges and aromatic interactions. The affinity for calcium is also almost three orders of magnitude lower than that of mesophilic subtilisin and the interactions with the solvent are significantly higher thanks to a large increase in the number of Asp residues in the loops connecting secondary structures. The relation between flexibility and activity was investigated by site-directed mutagenesis tending mainly to increase the rigidity of the molecular edifice through the incorporation of additional salt bridge, disulfide bridge, aromatic interaction and by increasing the affinity of the enzyme for calcium. An important stabilization of the molecular structure was achieved through a modification of a calcium ligand T85D. The thermostability of the mutated product expressed in a mesophilic Bacillus reaches that of mesophilic subtilisin. Most important is the fact that this mutation further enhances the specific activity by a factor close to 2 when compared to the wild type enzyme so that the overall activity of the mutated cold enzyme is about 20 times higher than that of mesophilic subtilisin, illustrating the fact that thermostability is not systematically inversely related to specific activity. This opens new perspectives in the use of cold enzymes in biotechnology.