Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

Metalloproteinase-1 (MMP-1) catalyzed collagen degradation is essential for a wide variety of normal physiological processes, while at the same time contributing to several diseases in humans. Therefore, a comprehensive understanding of this process is of great importance. Although crystallographic and spectroscopic studies provided fundamental information about the structure and function of MMP-1, the precise mechanism of collagen degradation especially considering the complex and flexible structure of the substrate, remains poorly understood. In addition, how the protein environment dynamically reorganizes at the atomic scale into a catalytically active state capable of collagen hydrolysis remains unknown. In this study, we applied experimentally-guided multiscale molecular modeling methods including classical molecular dynamics (MD), well-tempered (WT) classical metadynamics (MetD), combined quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM MetD simulations to explore and characterize the early catalytic events of MMP-1 collagenolysis. Importantly the study provided a complete atomic and dynamic description of the transition from the open to the closed form of the MMP-1•THP complex. Notably, the formation of catalytically active Michaelis complex competent for collagen cleavage was characterized. The study identified the changes in the coordination state of the catalytic zinc(II) associated with the conformational transformation and the formation of catalytically productive ES complex. Our results confirm the essential role of the MMP-1 catalytic domain's α-helices (hA, hB and hC) and the linker region in the transition to the catalytically competent ES complex. Overall, the results provide unique mechanistic insight into the conformational transformations and associated changes in the coordination state of the catalytic zinc(II) that would be important for the design of effective MMP-1 inhibitors.

Unraveling Binding Mechanism and Stability of Urease Inhibitors: A QM/MM MD Study.

Soil bacteria can produce urease, which catalyzes the hydrolysis of urea to ammonia (NH3) and carbamate. A variety of urease inhibitors have been proposed to reduce NH3 volatilization by interfering with the urease activity. We report a quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) study on the mechanism employed for the inhibition of urease by three representative competitive inhibitors; namely, acetohydroxamic acid (AHA), hydroxyurea (HU), and N-(n-butyl)phosphorictriamide (NBPTO). The possible connections between the structural and thermodynamical properties and the experimentally observed inhibition efficiency were evaluated and characterized. We demonstrate that the binding affinity decreases in the order NBPTO >> AHA > HU in terms of the computed activation and reaction free energies. This trend also indicates that NBPTO shows the highest inhibitory activity and the lowest IC50 value of 2.1 nM, followed by AHA (42 µM) and HU (100 µM). It was also found that the X=O moiety (X = carbon or phosphorous) plays a crucial role in the inhibitor binding process. These findings not only elucidate why the potent urease inhibitors are effective but also have implications for the design of new inhibitors.

Exploring Dihydroflavonol-4-Reductase Reactivity and Selectivity by QM/MM-MD Simulations.

Flavonoids are secondary metabolites ubiquitously found in plants. Their antioxidant properties make them highly interesting natural compounds for use in pharmacology. Therefore, unravelling the mechanisms of flavonoid biosynthesis is an important challenge. Among all the enzymes involved in this biosynthetic pathway, dihydroflavonol-4-reductase (DFR) plays a key role in the production of anthocyanins and proanthocyanidins. Here, we provide new information on the mechanism of action of this enzyme by using QM/MM-MD simulations applied to both dihydroquercetin (DHQ) and dihydrokaempferol (DHK) substrates. The consideration of these very similar compounds shed light on the major role played by the enzyme on the stabilization of the transition state but also on the activation of the substrate before the reaction through near-attack conformer effects.

Quantum chemistry study of the multiphoton absorption in enhanced green fluorescent protein at the single amino acid residue level.

The chromophore (CRO) of fluorescent proteins (FPs) is embedded in a complex environment that is a source of specific interactions with the CRO. Understanding how these interactions influence FPs spectral properties is important for a directed design of novel markers with desired characteristics. In this work, we apply computational chemistry methods to gain insight into one-, two- and three-photon absorption (1PA, 2PA, 3PA) tuning in enhanced green fluorescent protein (EGFP). To achieve this goal, we built EGFP models differing in: i) number and position of hydrogen-bonds (h-bonds) donors to the CRO and ii) the electric field, as approximated by polarizable force field, acting on the CRO. We find that h-bonding to the CRO's phenolate oxygen results in stronger one- and multiphoton absorption. The brighter absorption can be also achieved by creating more positive electric field near the CRO's phenolate moiety. Interestingly, while individual CRO - environment h-bonds usually enhance 1PA and 2PA, it takes a few h-bond donors to enhance 3PA. Clearly, response of the absorption intensity to many-body effects depends on the excitation mechanism. We further employ symmetry-adapted perturbation theory (SAPT) to reveal excellent (2PA) and good (3PA) correlation of multiphoton intensity with electrostatic and induction interaction energies. This points to importance of accounting for mutual CRO - environment polarization in quantitative calculations of absorption spectra in FPs.

Spin-projected QM/MM Free Energy Simulations for Oxidation Reaction of Guanine in B-DNA by Singlet Oxygen.

Guanine is the most susceptible base to oxidation damage induced by reactive oxygen species including singlet oxygen (1 O2 , 1 Δg ). We clarify whether the first step of guanine oxidation in B-DNA proceeds via either a zwitterionic or a diradical intermediate. The free energy profiles are calculated by means of a combined quantum mechanical and molecular mechanical (QM/MM) method coupled with the adaptive biasing force (ABF) method. To describe the open-shell electronic structure of 1 O2 correctly, the broken-symmetry spin-unrestricted density functional theory (BS-UDFT) with an approximate spin projection (AP) correction is applied to the QM region. We find that the effect of spin contamination on the activation and reaction free energies is up to ∼8 kcal mol-1 , which is too large to be neglected. The QM(AP-ULC-BLYP)/MM-based free energy calculations also reveal that the reaction proceeds through a diradical transition state, followed by a conversion to a zwitterionic intermediate. Our computed activation energy of 5.2 kcal mol-1 matches experimentally observed range (0∼6 kcal mol-1 ).

Origin and functional differentiation of (E)-ß-ocimene synthases reflect the expansion of monoterpenes in angiosperms.

The acquisition of new metabolic activities is a major force driving evolution. We explored, from the perspectives of gene family expansion and the evolutionary adaptability of proteins, how new functions have arisen in which terpene synthases diverged. Monoterpenoids are diverse natural compounds that can be divided into cyclic and acyclic skeleton forms according to their chemical structure. We demonstrate, through phylogenetic reconstructions and genome synteny analyses, that the (E)-ß-ocimene synthases, which are acyclic monoterpene synthases (mTPSs), appear to have arisen several times in independent lineages during plant evolution. Bioinformatics analyses and classical mutation experiments identified four sites (I388, F420, S446, and F485) playing important roles in the neofunctionalization of mTPSs. Incubation of neryl diphosphate with Salvia officinalis 1,8-cineole synthase (SCS) and mutated proteins show that these four sites obstruct the isomerization of geranyl diphosphate. Quantum mechanical/molecular mechanical molecular dynamics simulations of models of SCS, SCSY420F/I446S, and SCSN338I/Y420F/I446S/L485F with (3R)-linalyl diphosphate suggest that mutations changed the configuration of the intermediate to obtain new activities. These results provide new perspectives on the evolution of mTPSs, explain the convergent evolution of (E)-ß-ocimene synthases at the molecular level, and identify key residues to control the specificity of engineered mTPSs.

Development of massive multilevel molecular dynamics simulation program, Platypus (PLATform for dYnamic Protein Unified Simulation), for the elucidation of protein functions.

A massively parallel program for quantum mechanical-molecular mechanical (QM/MM) molecular dynamics simulation, called Platypus (PLATform for dYnamic Protein Unified Simulation), was developed to elucidate protein functions. The speedup and the parallelization ratio of Platypus in the QM and QM/MM calculations were assessed for a bacteriochlorophyll dimer in the photosynthetic reaction center (DIMER) on the K computer, a massively parallel computer achieving 10 PetaFLOPs with 705,024 cores. Platypus exhibited the increase in speedup up to 20,000 core processors at the HF/cc-pVDZ and B3LYP/cc-pVDZ, and up to 10,000 core processors by the CASCI(16,16)/6-31G** calculations. We also performed excited QM/MM-MD simulations on the chromophore of Sirius (SIRIUS) in water. Sirius is a pH-insensitive and photo-stable ultramarine fluorescent protein. Platypus accelerated on-the-fly excited-state QM/MM-MD simulations for SIRIUS in water, using over 4000 core processors. In addition, it also succeeded in 50-ps (200,000-step) on-the-fly excited-state QM/MM-MD simulations for the SIRIUS in water.

Catalytic deAMPylation in AMPylation-inhibitory/assistant forms of FICD protein.

DeAMPylation, as a reversible reaction of AMPylation and mediated by the endoplasmic reticulum-localized enzyme FICD (filamentation induced by cAMP domain protein, also known as HYPE), is an important process in protein posttranslational modifications (PTMs). Elucidating the function and catalytic details of FICD is of vital importance to provide a comprehensive understanding of protein folding homeostasis. However, the detailed deAMPylation mechanism is still unclear. Furthermore, the role of a conserved glutamine (Glu234), that plays an inhibitory role in the AMPylation response, is still an open question in the deAMPylation process. In the present work, the elaborated deAMPylation mechanisms with AMPylation-inhibitory/assistant forms of FICD (wild type and Glu234Ala mutant) were investigated based on the QM(DFT)/MM MD approach. The results revealed that deAMPylation was triggered by proton transfer from protonated histidine (His363) to AMPylated threonine, instead of a nucleophilic attack of water molecules adding to the phosphorus of AMP. The free energy barrier of deAMPylation in the wild type (∼17.3 kcal/mol) is consistent with that in the Glu234Ala mutant of FICD (∼17.1 kcal/mol), suggesting that the alteration of the Glu234 residue does not affect the deAMPylation reaction and indirectly verifying the inducement of deAMPylation in FICD. In the wild type, the proton in the nucleophilic water molecule is transferred to Glu234, whereas it is delivered to Asp367 through the hydrogen-bond network of coordinated water molecules in the Glu234Ala mutant. The present findings were inspirational for understanding the catalytic and inhibitory mechanisms of FICD-mediated AMP transfer, paving the way for further studies on the physiological role of FICD protein.

Enzymatic Polymerization of PCL-PEG Co-polymers for Biomedical Applications.

Biodegradable polymers, obtained via chemical synthesis, are currently employed in a wide range of biomedical applications. However, enzymatic polymerization is an attractive alternative because it is more sustainable and safer. Many lipases can be employed in ring-opening polymerization (ROP) of biodegradable polymers. Nevertheless, the harsh conditions required in industrial context are not always compatible with their enzymatic activity. In this work, we have studied a thermophilic carboxylesterase and the commonly used Lipase B from Candida antarctica (CaLB) for tailored synthesis of amphiphilic polyesters for biomedical applications. We have conducted Molecular Dynamics (MD) and Quantum Mechanics/Molecular Mechanics (QM/MM) MD simulations of the synthesis of Polycaprolactone-Polyethylene Glycol (PCL-PEG) model co-polymers. Our insights about the reaction mechanisms are important for the design of customized enzymes capable to synthesize different polyesters for biomedical applications.

Experiment and Simulation Reveal How Mutations in Functional Plasticity Regions Guide Plant Monoterpene Synthase Product Outcome.

Monoterpenes (C10 isoprenoids) are a structurally diverse group of natural compounds that are attractive to industry as flavours and fragrances. Monoterpenes are produced from a single linear substrate, geranyl diphosphate, by a group of enzymes called the monoterpene cyclases/synthases (mTC/Ss) that catalyse high-energy cyclisation reactions involving unstable carbocation intermediates. Efforts towards producing monoterpenes via biocatalysis or metabolic engineering often result in the formation of multiple products due to the nature of the highly branched reaction mechanism of mTC/Ss. Rational engineering of mTC/Ss is hampered by the lack of correlation between the active site sequence and cyclisation type. We used available mutagenesis data to show that amino acids involved in product outcome are clustered and spatially conserved within the mTC/S family. Consensus sequences for three such plasticity regions were introduced in different mTC/S with increasingly complex cyclisation cascades, including the model enzyme limonene synthase (LimS). In all three mTC/S studied, mutations in the first two regions mostly give rise to products that result from premature quenching of the linalyl or α-terpinyl cations, suggesting that both plasticity regions are involved in the formation and stabilisation of cations early in the reaction cascade. A LimS variant with mutations in the second region (S454G, C457V, M458I), produced mainly more complex bicyclic products. QM/MM MD simulations reveal that the second cyclisation is not due to compression of the C2-C7 distance in the α-terpinyl cation, but is the result of an increased distance between C8 of the α-terpinyl cation and two putative bases (W324, H579) located on the other side of the active site, preventing early termination by deprotonation. Such insights into the impact of mutations can only be obtained using integrated experimental and computational approaches, and will aid the design of altered mTC/S activities towards clean monoterpenoid products.

Mathematical and Computational Techniques for Drug Discovery: Promises and Developments.

We review various mathematical and computational techniques for drug discovery exemplifying some recent works pertinent to group theory of nested structures of relevance to phylogeny, topological, computational and combinatorial methods for drug discovery for multiple viral infections. We have reviewed techniques from topology, combinatorics, graph theory and knot theory that facilitate topological and mathematical characterizations of protein-protein interactions, molecular-target interactions, proteomics, genomics and statistical data reduction procedures for a large set of starting chemicals in drug discovery. We have provided an overview of group theoretical techniques pertinent to phylogeny, protein dynamics especially in intrinsically disordered proteins, DNA base permutations and related algorithms. We consider computational techniques derived from high level quantum chemical computations such as QM/MM ONIOM methods, quantum chemical optimization of geometries complexes, and molecular dynamics methods for providing insights into protein-drug interactions. We have considered complexes pertinent to Hepatitis Virus C non-structural protein 5B polymerase receptor binding of C5-Arylidebne rhodanines, complexes of synthetic potential vaccine molecules with dengue virus (DENV) and HIV-1 virus as examples of various simulation studies that exemplify the utility of computational tools. It is demonstrated that these combinatorial and computational techniques in conjunction with experiments can provide promising new insights into drug discovery. These techniques also demonstrate the need to consider a new multiple site or allosteric binding approach to drug discovery, as these studies reveal the existence of multiple binding sites.

QM/MM MD and Free Energy Simulation Study of Methyl Transfer Processes Catalyzed by PKMTs and PRMTs.

Methyl transfer processes catalyzed by protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) control important biological events including transcriptional regulation and cell signaling. One important property of these enzymes is that different PKMTs and PRMTs catalyze the formation of different methylated product (product specificity). These different methylation states lead to different biological outcomes. Here, we review the results of quantum mechanics/molecular mechanics molecular dynamics and free energy simulations that have been performed to study the reaction mechanism of PKMTs and PRMTs and the mechanism underlying the product specificity of the methyl transfer processes.

PUPIL: A Software Integration System for Multi-Scale QM/MM-MD Simulations and Its Application to Biomolecular Systems.

PUPIL (Program for User Package Interfacing and Linking) implements a distinctive multi-scale approach to hybrid quantum mechanical/molecular mechanical molecular dynamics (QM/MM-MD) simulations. Originally developed to interface different external programs for multi-scale simulation with applications in the materials sciences, PUPIL is finding increasing use in the study of complex biological systems. Advanced MD techniques from the external packages can be applied readily to a hybrid QM/MM treatment in which the forces and energy for the QM region can be computed by any of the QM methods available in any of the other external packages. Here, we give a survey of PUPIL design philosophy, main features, and key implementation decisions, with an orientation to biomolecular simulation. We discuss recently implemented features which enable highly realistic simulations of complex biological systems which have more than one active site that must be treated concurrently. Examples are given.