Comparison of coupled motions in Escherichia coli and Bacillus subtilis dihydrofolate reductase

Hybrid quantum/classical molecular dynamics simulations are used to compare the role of protein motion in the hydride transfer reaction catalyzed by Escherichia coli and Bacillus subtilis dihydrofolate reductase (DHFR). These two enzymes have 44% sequence identity, and the experimentally determined structures and hydride transfer rates are similar. The simulations indicate that the tertiary structures of both enzymes evolve in a similar manner during the hydride transfer reaction. In both enzymes, the donor-acceptor distance decreases to approximately 2.7 Angstroms at the transition state configurations to enable hydride transfer. Zero point energy and hydrogen tunneling effects are found to be significant for both enzymes. Covariance and rank correlation analyses of motions throughout the protein and ligands illustrate that E. coli and B. subtilis DHFR exhibit both similarities and differences in the equilibrium fluctuations and the conformational changes along the collective reaction coordinate for hydride transfer. A common set of residues that play a significant role in the network of coupled motions leading to configurations conducive to hydride transfer for both E. coli and B. subtilis DHFR was identified. These results suggest a balance between conservation and flexibility in the thermal motions and conformational changes during hydride transfer. Homologous protein structures, in conjunction with conformational sampling, enable enzymes with different sequences to catalyze the same hydride transfer reaction with similar efficiency.     

A quantum mechanics/molecular mechanics study of the protein-ligand interaction of two potent inhibitors of human O-GlcNAcase: PUGNAc and NAG-thiazoline

O-glycoprotein 2-acetamino-2-deoxy-beta- d-glucopyranosidase ( O-GlcNAcase) hydrolyzes 2-acetamido-2-deoxy-beta- d-glucopyranose ( O-GlcNAc) residues of serine/threonine residues of modified proteins. O-GlcNAc is present in many intracellular proteins and appears to have a role in the etiology of several diseases including cancer, Alzheimer's disease, and type II diabetes. In this work, we have carried out molecular dynamics simulations using a hybrid quantum mechanics/molecular mechanics approach to determine the binding of two potent inhibitors, PUGNAc and NAG, with a bacterial O-GlcNAcase. The results of these simulations show that Asp-401, Asp-298, and Asp-297 residues play an important role in the protein-inhibitor interactions. These results might be useful to design compounds with more interesting inhibitory activity on the basis of its three-dimensional structure.     

Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC

The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)-2,3-oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in nature. Friedelin has a unique PT skeleton involving a fascinating nine-step cation shuttle run (CSR) cascade rearrangement reaction, in which the carbocation formed at C2 moves to the other side of the skeleton, runs back to C3 to yield a friedelin cation, which is finally deprotonated. However, as crystal structure data of plant OSCs are lacking, it remains unknown why the CSR cascade reactions occur in friedelin biosynthesis, as does the exact catalytic mechanism of the CSR. In this study, we determined the first cryogenic electron microscopy structure of a plant OSC, friedelin synthase, from Tripterygium wilfordii Hook. f (TwOSC). We also performed quantum mechanics/molecular mechanics simulations to reveal the energy profile for the CSR cascade reaction and identify key residues crucial for PT skeleton formation. Furthermore, we semirationally designed two TwOSC mutants, which significantly improved the yields of friedelin and β-amyrin, respectively.     

Elucidating collision induced dissociation products and reaction mechanisms of protonated uracil by coupling chemical dynamics simulations with tandem mass spectrometry experiments

In this study we have coupled mixed quantum‐classical (quantum mechanics/molecular mechanics) direct chemical dynamics simulations with electrospray ionization/tandem mass spectrometry experiments in order to achieve a deeper understanding of the fragmentation mechanisms occurring during the collision induced dissociation of gaseous protonated uracil. Using this approach, we were able to successfully characterize the fragmentation pathways corresponding to ammonia loss (m/z 96), water loss (m/z 95) and cyanic or isocyanic acid loss (m/z 70). Furthermore, we also performed experiments with isotopic labeling completing the fragmentation picture. Remarkably, fragmentation mechanisms obtained from chemical dynamics simulations are consistent with those deduced from isotopic labeling. Copyright © 2015 John Wiley & Sons, Ltd.     

Microsecond timescale MD simulations at the transition state of PmHMGR predict remote allosteric residues

Understanding the mechanisms of enzymatic catalysis requires a detailed understanding of the complex interplay of structure and dynamics of large systems that is a challenge for both experimental and computational approaches. More importantly, the computational demands of QM/MM simulations mean that the dynamics of the reaction can only be considered on a timescale of nanoseconds even though the conformational changes needed to reach the catalytically active state happen on a much slower timescale. Here we demonstrate an alternative approach that uses transition state force fields (TSFFs) derived by the quantum-guided molecular mechanics (Q2MM) method that provides a consistent treatment of the entire system at the classical molecular mechanics level and allows simulations at the microsecond timescale. Application of this approach to the second hydride transfer transition state of HMG-CoA reductase from Pseudomonas mevalonii (PmHMGR) identified three remote residues, R396, E399 and L407, (15–27 Å away from the active site) that have a remote dynamic effect on enzyme activity. The predictions were subsequently validated experimentally via site-directed mutagenesis. These results show that microsecond timescale MD simulations of transition states are possible and can predict rather than just rationalize remote allosteric residues.

Transition state force fields enable MD simulations at the transition state of HMGCoA reductase that sample the transition state ensemble on the μs timescale to identify remote residues that affect the reaction rate.     

A molecular model of the folate binding site of Pneumocystis carinii dihydrofolate reductase

Summary The inhibition of Pneumocystis carinii dihydrofolate reductase (DHFR) continues to be the major treatment strategy for P. carinii pneumonia (PCP). The design of new anti-pneumocystis agents would be significantly enhanced by the availability of a 3D model of the methotrexate (MTX) binding site of the P. carinii DHFR. However, an X-ray crystal structure of the P. carinii DHFR is not yet available. Alignment of the amino acid sequences of P. carinii and Lactobacillus casei DHFRs indicates that the two proteins show approximately 80% homology among MTX binding-site residues. This high level of homology suggests that the L. casei DHFR MTX binding-site structure could serve as a structural template in developing a model of the P. carinii DHFR MTX binding site. Therefore, the X-ray crystal structure of L. casei DHFR was used to develop a 3D model of the methotrexate binding site of P. carinii DHFR. The molecular modeling and dynamics software QUANTA/CHARMm was used. Amino acid residue mutations and deletions were performed using QUANTA and macromolecular minimizations were achieved with CHARMm. The MTX binding-site residues of L. casei DHFR were mutated to the corresponding residues of the P. carinii DHFR sequence. The resulting structure was extensively minimized. The resulting P. carinii MTX binding-site model showed significant differences in hydrogen-bonding patterns from the L. casei MTX binding site. Also, the P. carinii site is more hydrophobic than the corresponding L. casei site. Analysis of atom-to-atom close contacts between methotrexate and protein binding-site residues indicates that the P. carinii MTX binding-site complex is primarily stabilized by hydrophobic interactions, while the L. casei complex is mostly stabilized by electrostatic interactions. The model is consistent with the observed increased sensitivity of P. carinii DHFR to lipid-soluble inhibitors and provides a rational basis for the design of new anti-pneumocystis agents.     

Substrate‐Guided Front‐Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N‐Acetylgalactosaminyltransferase 2

The retaining glycosyltransferase GalNAc‐T2 is a member of a large family of human polypeptide GalNAc‐transferases that is responsible for the post‐translational modification of many cell‐surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum‐mechanics/molecular‐mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic‐electronic level of detail. Our study provides a detailed structural rationale for an ordered bi–bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front‐face S_Ni‐type reaction in which the substrate N‐acetyl sugar substituent coordinates efficient glycosyl transfer.     

Dioxygen Binding in the Active Site of Histone Demethylase JMJD2A and the Role of the Protein Environment

JMJD2A catalyses the demethylation of di‐ and trimethylated lysine residues in histone tails and is a target for the development of new anticancer medicines. Mechanistic details of demethylation are yet to be elucidated and are important for the understanding of epigenetic processes. We have evaluated the initial step of histone demethylation by JMJD2A and demonstrate the dramatic effect of the protein environment upon oxygen binding using quantum mechanics/molecular mechanics (QM/MM) calculations. The changes in electronic structure have been studied for possible spin states and different conformations of O₂, using a combination of quantum and classical simulations. O₂ binding to this histone demethylase is computed to occur preferentially as an end‐on superoxo radical bound to a high‐spin ferric centre, yielding an overall quintet ground state. The favourability of binding is strongly influenced by the surrounding protein: we have quantified this effect using an energy decomposition scheme into electrostatic and dispersion contributions. His182 and the methylated lysine assist while Glu184 and the oxoglutarate cofactor are deleterious for O₂ binding. Charge separation in the superoxo‐intermediate benefits from the electrostatic stabilization provided by the surrounding residues, stabilizing the binding process significantly. This work demonstrates the importance of the extended protein environment in oxygen binding, and the role of energy decomposition in understanding the physical origin of binding/recognition.     

Theoretical Insights into Chirality‐Controlled SWCNT Growth from a Cycloparaphenylene Template

A self‐assembly mechanism for low‐temperature SWCNT growth from a [6]cycloparaphenylene ([6]CPP) precursor via ethynyl (C₂H) radical addition is presented, based on non‐equilibrium quantum chemical molecular dynamics (QM/MD) simulations and density functional theory (DFT) calculations. This mechanism, which maintains the (6,6) armchair chirality of a SWCNT fragment throughout the growth process, is energetically more favorable than a previously proposed Diels–Alder‐based growth mechanisms [E. H. Fort, et al., J. Mater. Chem. 2011 , 21, 1373]. QM/MD simulations and DFT calculations show that C₂H radicals play dual roles during SWCNT growth, by abstracting hydrogen from the SWCNT fragment and providing the carbon source necessary for growth itself. Simulations demonstrate that chirality‐controlled SWCNT growth from macrocyclic hydrocarbon seed molecules with pre‐selected edge structure can be accomplished when the reaction conditions are carefully selected for hydrogen abstraction by radical species during the growth process.     

A quantum mechanics/molecular mechanics study of the protein-ligand interaction for inhibitors of HIV-1 integrase

Human immunodeficiency virus type-1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Diketo acids such as L-731,988 and S-1360 are potent and selective inhibitors of HIV-1 IN. In this study, we used molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy between HIV-1 IN and L-731,988 and 10 of its derivatives and analogues. This hybrid methodology has the advantage that it includes quantum effects such as ligand polarisation upon binding, which can be very important when highly polarisable groups are embedded in anisotropic environments, as for example in metal-containing active sites. Furthermore, an energy decomposition analysis was performed to determine the contributions of individual residues to the enzyme-inhibitor interactions on averaged structures obtained from rather extensive conformational sampling. Analysis of the results reveals first that there is a correlation between protein-ligand interaction energy and experimental strand transfer into human chromosomes and secondly that the Asn-155, Lys-156 and Lys-159 residues and the Mg(2+) ion are crucial to anti-HIV IN activity. These results may explain the available experimental data.     

Selectivity analysis of 5-(arylthio)-2,4-diaminoquinazolines as inhibitors of Candida albicans dihydrofolate reductase by molecular dynamics simulations

A series of 5-(arylthio)-2,4-diaminoquinazolines are known as selective inhibitors of dihydrofolate reductase (DHFR) from Candida albicans. We have performed docking and molecular dynamics simulations of these inhibitors with C. albicans and human DHFR to understand the basis for selectivity of these agents. Study was performed on a selected set of 10 compounds with variation in structure and activity. Molecular dynamics simulations were performed at 300 K for 45 ps with equilibration for 10 ps. Trajectory data was analyzed on the basis of hydrogen bond interactions, energy of binding and conformational energy difference. The results indicate that hydrogen bonds formed between the compound and the active site residues are responsible for inhibition and higher potency. The selectivity index, i.e the ratio of I₅₀ against human DHFR to I₅₀ against fungal DHFR, is mainly determined by the conformation adapted by the compounds within the active site of two enzymes. Since the human DHFR active site is rigid, the compound is trapped in a higher energy conformation. This energy difference between the two conformations E mainly governs the selectivity against fungal DHFR. The information generated from this analysis of potency and selectivity should be useful for further work in the area of antifungal research.     

Steric Effects Govern the Photoactivation of Phytochromes

Phytochromes constitute a superfamily of photoreceptor proteins existing in two forms that absorb red (Pr) and far‐red (Pfr) light. Although it is well‐known that the conversion of Pr into Pfr (the biologically active form) is triggered by a Z→E photoisomerization of the linear tetrapyrrole chromophore, direct evidence is scarce as to why this reaction always occurs at the methine bridge between pyrrole rings C and D. Here, we present hybrid quantum mechanics/molecular mechanics calculations based on a high‐resolution Pr crystal structure of Deinococcus radiodurans bacteriophytochrome to investigate the competition between all possible photoisomerizations at the three different (AB, BC and CD) methine bridges. The results demonstrate that steric interactions with the protein are a key discriminator between the different reaction channels. In particular, it is found that such interactions render photoisomerizations at the AB and BC bridges much less probable than photoisomerization at the CD bridge.     

Parameters for molecular dynamics simulations of iron‐sulfur proteins

Iron‐sulfur proteins involved in electron transfer reactions have finely tuned redox potentials, which allow them to be highly efficient and specific. Factors such as metal center solvent exposure, interaction with charged residues, or hydrogen bonds between the ligand residues and amide backbone groups have all been pointed out to cause such specific redox potentials. Here, we derived parameters compatible with the AMBER force field for the metal centers of iron‐sulfur proteins and applied them in the molecular dynamics simulations of three iron‐sulfur proteins. We used density‐functional theory (DFT) calculations and Seminario's method for the parameterization. Parameter validation was obtained by matching structures and normal frequencies at the quantum mechanics and molecular mechanics levels of theory. Having guaranteed a correct representation of the protein coordination spheres, the amide H‐bonds and the water exposure to the ligands were analyzed. Our results for the pattern of interactions with the metal centers are consistent to those obtained by nuclear magnetic resonance spectroscopy (NMR) experiments and DFT calculations, allowing the application of molecular dynamics to the study of those proteins. © 2013 Wiley Periodicals, Inc.     

酶催化过程的全程模拟

酶催化包括底物到活性区的输运、选择催化化学反应及产物释放等复杂过程,由于复杂的蛋白质环境效应,任一化学和非化学过程都有可能是决定酶活性的关键步骤。为了全面认识酶催化活性,我们对几类酶催化过程进行了广泛的组合量子/分子力学(QM/MM)和经典分子力学(MM)动力学模拟(MD)研究,详细地讨论了整个酶催化过程的分子机制、关键残基的作用和蛋白质环境效应,丰富了对酶催化活性的认识。随着多尺度模型和计算模拟方法的进一步完善与发展,有望实现超大复杂生物酶催化过程的全程模拟研究,为酶工程领域的相关研究提供支持。     

Freezing a single distal motion in dihydrofolate reductase

Constraining a single motion between distal residues separated by approximately 28 A in hybrid quantum/classical molecular dynamics simulations is found to increase the free energy barrier for hydride transfer in dihydrofolate reductase by approximately 3 kcal/mol. Our analysis indicates that a single distal constraint alters equilibrium motions throughout the enzyme on a wide range of time scales. This alteration of the conformational sampling of the entire system is sufficient to significantly increase the free energy barrier and decrease the rate of hydride transfer. Despite the changes in conformational sampling introduced by the constraint, the system assumes a similar transition state conformation with a donor-acceptor distance of approximately 2.72 A to enable the hydride transfer reaction. The modified thermal sampling leads to a substantial increase in the average donor-acceptor distance for the reactant state, however, thereby decreasing the probability of sampling the transition state conformations with the shorter distances required for hydride transfer. These simulations indicate that fast thermal fluctuations of the enzyme, substrate, and cofactor lead to conformational sampling of configurations that facilitate hydride transfer. The fast thermal motions are in equilibrium as the reaction progresses along the collective reaction coordinate, and the overall average equilibrium conformational changes occur on the slower time scale measured experimentally. Recent single molecule experiments suggest that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time scale of the hydride transfer reaction. Thus, introducing a constraint that modifies the conformational sampling of an enzyme could significantly impact its catalytic activity.     

On the Regio- and Stereospecificity of Arachidonic Acid Peroxidation Catalyzed by Mammalian 15-Lypoxygenases: A Combined Molecular Dynamics and QM/MM Study

15-Lipoxygenases (15-LOs) catalyse the peroxidation reaction of arachidonic acid (AA) in mammals with remarkable regio- and stereospecificity. This positional-specific peroxidation is of paramount importance because it determines the nature and biological functions of the final metabolites generated by each LO as a result of the oxidative metabolism of AA. Although several hypotheses have been formulated concerning the regio- and stereospecificity of LOs, the molecular basis of such behaviour is still unclear. Herein, we combined quantum mechanics/molecular mechanics calculations with molecular dynamics simulations of the complete rabbit 15-LO/AA solvated model to examine the most accepted hypotheses for the regio- and stereospecificity of LOs. We have found that the clue to explain this specificity is the oxygen-targeting hypothesis through steric shielding of specific residues (mainly Leu597, Gln548 and Phe175, as well as the AA tail itself). Our deductions are based primarily on the analysis of the energy barrier heights from the oxygen addition reaction profiles.     

To be or not to be butterfly: New mechanistic insights in the Aza‐Michael asymmetric addition of lithium (R)‐N‐benzyl‐N‐(α‐methylbenzyl)amide

The asymmetric Aza‐Michael addition of homochiral lithium benzylamides to α,β‐unsaturated esters represents an extended protocol to obtain enantioenriched β‐amino esters. An exhaustive mechanistic revision of the originally proposed mechanism is reported, developing a quantum mechanics/molecular mechanics protocol for the asymmetric Aza‐Michael reaction of homochiral lithium benzylamides. Explicit and implicit solvent schemes were considered, together with a proper account of long‐range dispersion forces, evaluated through a density functional theory benchmark of different functionals. Theoretical results showed that the diastereoselectivity is mainly controlled by the N‐α‐methylbenzyl moiety placing, deriving a Si/Re 99:1 diastereoselective ratio, in good agreement with reported experimental results. The main transition state geometries are two transition state conformers in a “V‐stacked” orientation of the amide's phenyl rings, differing in the tetrahydrofuran molecule arrangement coordinated to the metal center. Extensive conformational sampling and quantum‐level refinement give reasonable good speed/accuracy results, allowing this protocol to be extended to other similar Aza‐Michael reaction systems. © 2014 Wiley Periodicals, Inc.