Protein-ligand docking is currently an important tool in drug discovery efforts and an active area of research that has been the subject of important developments over the last decade. These are well portrayed in the rising number of available protein-ligand docking software programs, increasing level of sophistication of its most recent applications, and growing number of users. While starting by summarizing the key concepts in protein-ligand docking, this article presents an analysis of the evolution of this important field of research over the past decade. Particular attention is given to the massive range of alternatives, in terms of protein-ligand docking software programs currently available. The emerging trends in this field are the subject of special attention, while old established docking alternatives are critically revisited. Current challenges in the field of protein-ligand docking such as the treatment of protein flexibility, the presence of structural water molecules and its effect in docking, and the entropy of binding are dissected and discussed, trying to anticipate the next years in the field.

Quantum mechanics/molecular mechanics (QM/MM) methods offer a very appealing option for the computational study of enzymatic reaction mechanisms, by separating the problem into two parts that can be treated with different computational methods. Hence, in a QM/MM formalism, the part of the system in which catalysis actually occurs and that involves the active site, substrates and directly participating amino acid residues is treated at an adequate quantum mechanical level to describe the chemistry taking place. For the remaining of the enzyme, which does not participate directly in the reaction, but that typically involves a much larger number of atoms, molecular mechanics is employed, traditionally through the application of a biomolecular force field. When applied with care, QM/MM methods can be used with great advantage in comparing, at a structural and energetic level, different mechanistic proposals, discarding mechanistic alternatives and proposing new mechanistic pathways that are consistent with the available experimental data. With time, diverse flavors within the QM/MM methods have emerged, differing in a variety of technical and conceptual aspects. Hence present alternatives differ between additive and subtractive QM/MM schemes, the type of boundary schemes, and the way in which the electrostatic interactions between the two regions are accounted for. Also, single‐conformation QM/MM, multi‐PES approaches, and QM/MM Molecular Dynamics coexist today, each type with its own advantages and limitations. This review focuses on the application of QM/MM methods in the study of enzymatic reaction mechanisms, briefly presenting also the most important technical aspects involved in these calculations. Particular attention is dedicated to the application of the single‐conformation QM/MM, multi‐PES QM/MM studies, and QM/MM‐FEP methods and to the advantages and disadvantages of the different types of QM/MM. Recent breakthroughs are also introduced. A selection of hand‐picked examples is used to illustrate such features. WIREs Comput Mol Sci 2017, 7:e1281. doi: 10.1002/wcms.1281 This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics Structure and Mechanism > Reaction Mechanisms and Catalysis Electronic Structure Theory > Combined QM/MM Methods

Polyethylene terephthalate (PET) has been widely used to make disposable bottles, among others, leading to massive PET waste accumulation in the environment. The discovery of the Ideonella sakaiensis PETase and MHETase enzymes, which hydrolyze PET into its constituting monomers, opened the possibility of a promising route for PET biorecycling. We describe an atomistic and thermodynamic interpretation of the catalytic reaction mechanism of PETase using umbrella sampling simulations at the robust PBE/MM MD level with a large QM region. The reaction mechanism takes place in two stages, acylation and deacylation, each of which occurs through a single, associative, concerted and asynchronous step. Acylation consists of proton transfer from Ser131 to His208, concerted with a nucleophilic attack by Ser131 on the substrate, leading to a tetrahedral transition state, which subsequently results in the release of MHET after the breaking of the ester bond. Deacylation is driven by deprotonation of an active site water molecule by His208, with the resulting hydroxide attacking the acylated Ser131 intermediate and breaking its bond to the substrate. Subsequently, His208 transfers the water proton to Ser131, with ensuant formation of MHET and enzyme regeneration. The rate-limiting acylation has a free energy barrier of 20.0 kcal·mol–1, consistent with the range of experimental values of 18.0–18.7 kcal·mol–1. Finally, we identify residues whose mutation should increase the enzyme turnover. Specifically, mutation of Asp83, Asp89, and Asp157 by nonpositive residues is expected to decrease the barrier of the rate-limiting step. This work led to the understanding of the catalytic mechanism of PETase and opened the way for additional rational enzyme engineering.

We present a new formulation to deal with the consistency problem of a massive spin-2 field in a curved spacetime. Using Fierz variables to represent the spin-2 field, we show how to avoid the arbitrariness and inconsistency that exists in the standard formulation of spin-2 field coupled to gravity. The superiority of the Fierz frame appears explicitly in the combined set of equations for spin-2 field and gravity: it preserves the standard Einstein equations of motion.

We show that the graviton has a mass in an anti-de Sitter (Λ < 0) background given by mΛ. This is precisely the fine-tuning value required for the perturbed gravitational field to mantain its two degrees of freedom.

Dehydratase (DH) is a catalytic domain of the mammalian fatty acid synthase (mFAS), a multidomain enzyme with seven different active sites that work in tandem to carry out the biosynthesis of palmitic acid for de novo lipogenesis. DH catalyzes the dehydration of the β-hydroxyacyl to an α,β-unsaturated acyl intermediate. We have conducted hybrid QM/MM calculations to clarify the catalytic mechanism for the DH domain at the ONIOM­(DFT/Amber) level of theory. The results have shown that the dehydration step occurs in two stages: (i) the His878-imidazole acts as a base deprotonating the Cα of the β-hydroxyacyl (HAC) substrate and (ii) the β-elimination of the β-hydroxyl of HAC proceeds with late protonation of the leaving hydroxide by the Asp1033-carboxylic group, forming a water molecule as a byproduct. The α-deprotonation depends on an oxyanion hole mechanism where the HAC’s α-carbonyl is anchored by two strong hydrogen bonds from the neighboring Gly888 and the intramolecular β-hydroxyl, positioning the Cα of HAC for deprotonation by His878. A positively charged His1037 improves the acidic character of Asp1033 and completes the catalytic triad in DH, because when His1037 is neutral the positively charged His878 behaves as the acid in the β-elimination step. We observe that the positively charged His1037 renders the β-elimination step more thermodynamically favorable (Δr G of −15.9 kcal·mol–1). The β-elimination step exhibits a Gibbs energy barrier of 14.1 kcal·mol–1 and it is the rate-limiting step of the reaction (in agreement with the experimental barrier of ∼17 kcal·mol–1. Nevertheless, the rate-limiting step does not seem to be dependent on the protonation of His1037. Through evaluation of the electrostatic effect per residue on the rate-limiting step, we concluded also that the electrostatic contribution of the enzyme’s body does not seem significant, even though there are many positively and negatively charged residues close to the leaving β-hydroxyl group of HAC.

A set of 92 density functionals was employed to accurately characterize thiol-disulfide exchange. The properties we have benchmarked throughout the study include the geometry of a 15 atoms model system, the potential energy surface, the activation barrier, and the energy of reaction for thiol-disulfide exchange. Reference energies were determined at the CCSD(T)/CBS//MP2/aug-cc-pVDZ level of theory, and reference geometries were calculated at the MP2/aug-cc-pVTZ level. M11-L, M06-2X, M06-HF, N12-SX, PBE1PBE, PBEh1PBE, and OHSE2PBE described better the geometry of the model system, with average deviations of 0.06 Å in bond lengths (0.06 Å in bond-breaking lengths) and 1.9° in bond angles. On the other hand, the potential energy surface and its gradient were more accurately described by the hybrid density functional BHandH, closely followed by mPW1N, mPW1K, and mPWB1K. The barrier height and energy of reaction were better reproduced by the BMK and M06-2X functionals (deviations of 0.17 and 0.07 kcal·mol(-1), respectively) for a set of 10 Pople's basis sets. MN12-SX and M11-L showed very good results for the widely used 6-311++G(2d,2p) basis set, with deviations of 0.02 and 0.05 kcal·mol(-1), respectively. We studied the effect of the split-valence, diffuse, and polarized functions in the activation barrier of thiol-disulfide exchange, for a set of 10 Pople's basis sets. While increasing the splitting and polarization may increase the activation barrier in approximately 1 kcal·mol(-1), diffuse functions generally contribute to decreasing it no more than 0.10 kcal·mol(-1). In general, 13 functionals provided energies within 1 kcal·mol(-1) of the reference value. The BB1K density functional is one of the best density functionals to characterize thiol-disulfide exchange reactions; however, several density functionals with modified Perdew-Wang exchange and about 40% Hartree-Fock exchange, such as mPW1K, mPW1N, and mPWB1K, show a good performance, too.

Human fatty acid synthase (hFAS) is a multifunctional enzyme involved in a wide diversity of biological functions. For instance, it is a precursor of phospholipids and other complex processes such as the de novo synthesis of long chain fatty acid. Human FAS is also a component of biological membranes and it is implicated in the overexpression of several types of cancers. In this work, we describe the catalytic mechanism of β-ketoreductase (KR), which is a catalytic domain of the hFAS enzyme that catalyzes the reduction of β-ketoacyl to β-hydroxyacyl with the concomitant oxidation of the NADPH cofactor. The catalysis by KR is an intermediate step in the cycle of reactions that elongate the substrate's carbon chain until the final product is obtained. We study and propose the catalytic mechanism of the KR domain determined using the hybrid QM/MM methodology, at the ONIOM(B3LYP/6-311+G(2d,2p):AMBER) level of theory. The results indicate that the reaction mechanism occurs in two stages: (i) nucleophilic attack by a NADPH hydride to the β-carbon of the substrate, together with an asynchronous deprotonation of the Tyr2034 by the oxygen of the β-alkoxide to hold the final alcohol product; and (ii) an asynchronous deprotonation of the hydroxyl in the NADP's ribose by Tyr2034, and of the Lys1995 by the resulting alkoxide in the former ribose to restore the protonation state of Tyr2034. The reduction step occurs with a Gibbs energy barrier of 11.7 kcal mol and a Gibbs reaction energy of -10.6 kcal mol. These results have provided an understanding of the catalytic mechanism of the KR hFAS domain, a piece of the heavy hFAS biosynthetic machinery.