Structural consequences of the active site substitution Cys181 ==> Ser in metallo-beta-lactamase from Bacteroides fragilis

The metallo-beta-lactamases require divalent cations such as zinc or cadmium for hydrolyzing the amide bond of beta-lactam antibiotics. The crystal structure of the Zn2+ -bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam bond of the substrate. It was previously reported that the replacement of the active site Cys181 by a serine residue severely impaired catalysis while atomic absorption measurements indicated that binding of the two zinc ions remained intact. Contradicting data emerge from recent mass spectrometry results, which show that only a single zinc ion binds to the C181S metallo-beta-lactamase. In the current study, the C181S mutant enzyme was examined at the atomic level by determining the crystal structure at 2.6 A resolution. The overall structure of the mutant enzyme is the same as that of the wild-type enzyme. At the mutation site, the side chain of Ser181 occupies the same position as that of the side chain of Cys181 in the wild-type protein. One zinc ion, Zn1, is present in the crystal structure; however, the site of the second zinc ion, Zn2 is unoccupied. A water molecule is associated with Zn1, reminiscent of the hydroxide seen in the structure of the wild-type enzyme but farther from the metal. The position of the water molecule is off the plane of the carboxylate group of Asp103; therefore, the water molecule may be less nucleophilic than a water molecule which is coplanar with the carboxylate group.     

High performance screening, structural and molecular dynamics analysis to identify H1 inhibitors from TCM Database@Taiwan

New-type oseltamivir-resistant H1N1 influenza viruses have been a major threat to human health since the 2009 flu pandemic. To resolve the drug resistance issue, we aimed to identify a new type of inhibitors against H1 from traditional Chinese medicine (TCM) by employing the world's largest TCM database () for virtual screening and molecular dynamics (MD). From the virtual screening results, sodium (+)-isolaricireinol-2 alpha-sulfate, sodium 3,4-dihydroxy-5-methoxybenzoic acid methyl ester-4-sulfate, sodium (E)-7-hydroxy-1,7-bis(4-hydroxyphenyl)hept-5-ene-3S-sulfonate, and 3-methoxytyramine-betaxanthin were identified as potential drug-like compounds. MD simulation of the binding poses with the key residues Asp103 and Glu83, as well as other binding site residues, identified higher numbers of hydrogen bonds than N-Acetyl-D-Glucosamine (NAG), the natural ligand of the esterase domain in H1. Ionic bonds, salt bridges, and electrostatic energy also contribute to binding stability. Key binding residues include Lys71, Glu83, Asp103, and Arg238. Structural moieties promoting H-bond or salt bridge formations at these locations greatly contribute to a stable ligand-protein complex. An available sodium atom for ionic interactions with Asp103 can further stabilize the ligands. Based on virtual screening, MD simulation, and interaction energy evaluation, TCM candidates demonstrate good potential as novel H1 inhibitors. In addition, the identified stabilizing features can provide insights for designing highly stable H1 inhibitors.     

Protonation state and substrate binding to B2 metallo-beta-lactamase CphA from Aeromonas hydrofila

The zinc enzymes metallo beta-lactamases counteract the beneficial action of beta-lactam antibiotics against bacterial infections, by hydrolyzing their beta-lactam rings. To understand structure/function relationships on a representative member of this class, the B2 M beta L CphA, we have investigated the H-bond pattern at the Zn enzymatic active site and substrate binding mode by molecular simulation methods. Extensive QM calculations at the DFT-BLYP level on eleven models of the protein active site, along with MD simulations of the protein in aqueous solution, allow us to propose two plausible protonation states for the unbound enzyme, which are probably in equilibrium. Docking procedures along with MD simulations and QM calculations suggest that in the complex between the enzyme and its substrate (biapenem), the latter is stable in only one of the two protonation states, in addition it exhibits two different binding modes, of which only one agrees with previous proposals. In both cases, the substrate is polarized as in aqueous solution. We conclude that addressing mechanistic issues on this class of enzymes requires a careful procedure to assign protonation states and substrate docking modes.     

Determinants of reactivity and selectivity in soluble epoxide hydrolase from quantum mechanics/molecular mechanics modeling

Soluble epoxide hydrolase (sEH) is an enzyme involved in drug metabolism that catalyzes the hydrolysis of epoxides to form their corresponding diols. sEH has a broad substrate range and shows high regio- and enantioselectivity for nucleophilic ring opening by Asp333. Epoxide hydrolases therefore have potential synthetic applications. We have used combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations (at the AM1/CHARMM22 level) and high-level ab initio (SCS-MP2) QM/MM calculations to analyze the reactions, and determinants of selectivity, for two substrates: trans-stilbene oxide (t-SO) and trans-diphenylpropene oxide (t-DPPO). The calculated free energy barriers from the QM/MM (AM1/CHARMM22) umbrella sampling MD simulations show a lower barrier for phenyl attack in t-DPPO, compared with that for benzylic attack, in agreement with experiment. Activation barriers in agreement with experimental rate constants are obtained only with the highest level of QM theory (SCS-MP2) used. Our results show that the selectivity of the ring-opening reaction is influenced by several factors, including proximity to the nucleophile, electronic stabilization of the transition state, and hydrogen bonding to two active site tyrosine residues. The protonation state of His523 during nucleophilic attack has also been investigated, and our results show that the protonated form is most consistent with experimental findings. The work presented here illustrates how determinants of selectivity can be identified from QM/MM simulations. These insights may also provide useful information for the design of novel catalysts for use in the synthesis of enantiopure compounds.     

A comparative study of trypsin specificity based on QM/MM molecular dynamics simulation and QM/MM GBSA calculation

Hydrogen bonding and polar interactions play a key role in identification of protein-inhibitor binding specificity. Quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations combined with DFT and semi-empirical Hamiltonian (AM1d, RM1, PM3, and PM6) methods were performed to study the hydrogen bonding and polar interactions of two inhibitors BEN and BEN1 with trypsin. The results show that the accuracy of treating the hydrogen bonding and polar interactions using QM/MM MD simulation of PM6 can reach the one obtained by the DFT QM/MM MD simulation. Quantum mechanics/molecular mechanics generalized Born surface area (QM/MM-GBSA) method was applied to calculate binding affinities of inhibitors to trypsin and the results suggest that the accuracy of binding affinity prediction can be significantly affected by the accurate treatment of the hydrogen bonding and polar interactions. In addition, the calculated results also reveal the binding specificity of trypsin: (1) the amidinium groups of two inhibitors generate favorable salt bridge interaction with Asp189 and form hydrogen bonding interactions with Ser190 and Gly214, (2) the phenyl of inhibitors can produce favorable van der Waals interactions with the residues His58, Cys191, Gln192, Trp211, Gly212, and Cys215. This systematic and comparative study can provide guidance for the choice of QM/MM MD methods and the designs of new potent inhibitors targeting trypsin.     

Lys169 of Human Glucokinase Is a Determinant for Glucose Phosphorylation: Implication for the Atomic Mechanism of Glucokinase Catalysis

Glucokinase (GK), a glucose sensor, maintains plasma glucose homeostasis via phosphorylation of glucose and is a potential therapeutic target for treating maturity-onset diabetes of the young (MODY) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). To characterize the catalytic mechanism of glucose phosphorylation by GK, we combined molecular modeling, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, experimental mutagenesis and enzymatic kinetic analysis on both wild-type and mutated GK. Our three-dimensional (3D) model of the GK-Mg²⁺-ATP-glucose (GMAG) complex, is in agreement with a large number of mutagenesis data, and elucidates atomic information of the catalytic site in GK for glucose phosphorylation. A 10-ns MD simulation of the GMAG complex revealed that Lys169 plays a dominant role in glucose phosphorylation. This prediction was verified by experimental mutagenesis of GK (K169A) and enzymatic kinetic analyses of glucose phosphorylation. QM/MM calculations were further used to study the role of Lys169 in the catalytic mechanism of the glucose phosphorylation and we found that Lys169 enhances the binding of GK with both ATP and glucose by serving as a bridge between ATP and glucose. More importantly, Lys169 directly participates in the glucose phosphorylation as a general acid catalyst. Our findings provide mechanistic details of glucose phorphorylation catalyzed by GK, and are important for understanding the pathogenic mechanism of MODY.     

Binding free energies for nicotine analogs inhibiting cytochrome P450 2A6 by a combined use of molecular dynamics simulations and QM/MM-PBSA calculations

Molecular dynamics (MD) simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations have been performed to explore the dynamic behaviors of cytochrome P450 2A6 (CYP2A6) binding with nicotine analogs (that are typical inhibitors) and to calculate their binding free energies in combination with Poisson–Boltzmann surface area (PBSA) calculations. The combined MD simulations and QM/MM-PBSA calculations reveal that the most important structural parameters affecting the CYP2A6-inhibitor binding affinity are two crucial internuclear distances, that is, the distance between the heme iron atom of CYP2A6 and the coordinating atom of the inhibitor, and the hydrogen-bonding distance between the N297 side chain of CYP2A6 and the pyridine nitrogen of the inhibitor. The combined MD simulations and QM/MM-PBSA calculations have led to dynamic CYP2A6-inhibitor binding structures that are consistent with the observed dynamic behaviors and structural features of CYP2A6-inhibitor binding, and led to the binding free energies that are in good agreement with the experimentally-derived binding free energies. The agreement between the calculated binding free energies and the experimentally-derived binding free energies suggests that the combined MD and QM/MM-PBSA approach may be used as a valuable tool to accurately predict the CYP2A6-inhibitor binding affinities in future computational design of new, potent and selective CYP2A6 inhibitors.     

Protonation states of intermediates in the reaction mechanism of [NiFe] hydrogenase studied by computational methods

The [NiFe] hydrogenases catalyse the reversible conversion of H₂ to protons and electrons. The active site consists of a Fe ion with one carbon monoxide, two cyanide, and two cysteine (Cys) ligands. The latter two bridge to a Ni ion, which has two additional terminal Cys ligands. It has been suggested that one of the Cys residues is protonated during the reaction mechanism. We have used combined quantum mechanical and molecular mechanics (QM/MM) geometry optimisations, large QM calculations with 817 atoms, and QM/MM free energy simulations, using the TPSS and B3LYP methods with basis sets extrapolated to the quadruple zeta level to determine which of the four Cys residues is more favourable to protonate for four putative states in the reaction mechanism, Ni-SI_a, Ni-R, Ni-C, and Ni-L. The calculations show that for all states, the terminal Cys-546 residue is most easily protonated by 14–51 kJ/mol, owing to a more favourable hydrogen-bond pattern around this residue in the protein.     

Dipeptide hydrolysis by the dinuclear zinc enzyme human renal dipeptidase: Mechanistic insights from DFT calculations

The reaction mechanism of the dinuclear zinc enzyme human renal dipeptidase is investigated using hybrid density functional theory. This enzyme catalyzes the hydrolysis of dipeptides and β-lactam antibiotics. Two different protonation states in which the important active site residue Asp288 is either neutral or ionized were considered. In both cases, the bridging hydroxide is shown to be capable of performing the nucleophilic attack on the substrate carbonyl carbon from its bridging position, resulting in the formation of a tetrahedral intermediate. This step is followed by protonation of the dipeptide nitrogen, coupled with C-N bond cleavage. The calculations establish that both cases have quite feasible energy barriers. When the Asp288 is neutral, the hydrolytic reaction occurs with a large exothermicity. However, the reaction becomes very close to thermoneutral with an ionized Asp288. The two zinc ions are shown to play different roles in the reaction. Zn1 binds the amino group of the substrate, and Zn2 interacts with the carboxylate group of the substrate, helping in orienting it for the nucleophilic attack. In addition, Zn2 stabilizes the oxyanion of the tetrahedral intermediate, thereby facilitating the nucleophilic attack.     

Comparison of force fields to study the zinc-finger containing protein NPL4, a target for disulfiram in cancer therapy

Molecular dynamics (MD) simulations are a powerful approach to studying the structure and dynamics of proteins related to health and disease. Advances in the MD field allow modeling proteins with high accuracy. However, modeling metal ions and their interactions with proteins is still challenging. NPL4 is a zinc-binding protein and works as a cofactor for p97 to regulate protein homeostasis. NPL4 is of biomedical importance and has been proposed as the target of disulfiram, a drug recently repurposed for cancer treatment. Experimental studies proposed that the disulfiram metabolites, bis-(diethyldithiocarbamate)‑copper and cupric ions, induce NPL4 misfolding and aggregation. However, the molecular details of their interactions with NPL4 and consequent structural effects are still elusive. Here, biomolecular simulations can help to shed light on the related structural details. To apply MD simulations to NPL4 and its interaction with copper the first important step is identifying a suitable force field to describe the protein in its zinc-bound states. We examined different sets of non-bonded parameters because we want to study the misfolding mechanism and cannot rule out that the zinc may detach from the protein during the process and copper replaces it. We investigated the force-field ability to model the coordination geometry of the metal ions by comparing the results from MD simulations with optimized geometries from quantum mechanics (QM) calculations using model systems of NPL4. Furthermore, we investigated the performance of a force field including bonded parameters to treat copper ions in NPL4 that we obtained based on QM calculations.     

Mutational analysis of metallo-beta-lactamase CcrA from Bacteroides fragilis

In an effort to evaluate the roles of Lys184, Asn193, and Asp103 in the binding and catalysis of metallo-beta-lactamase CcrA from Bacteroides fragilis, site-directed mutants of CcrA were generated and characterized using metal analyses, CD spectroscopy, and kinetic studies. Three Lys184 mutants were generated where the lysine was replaced with alanine, leucine, and glutamate, and the analysis of these mutants indicates that Lys184 is not greatly involved in binding of cephalosporins to CcrA; however, this residue does have a significant role in binding of penicillin G. Three Asn193 mutants were generated where the asparagine was replaced with alanine, leucine, and aspartate, and these mutants exhibited <4-fold decrease in k(cat), suggesting that Asn193 does not play a large role in catalysis. However, stopped-flow visible kinetic studies showed that the Asn193 mutants exhibit a slower substrate decay rate and no change in the product formation rate as compared with wild-type CcrA. These results support the proposed role of Asn193 in interacting with and activating substrate during catalysis. Two Asp103 mutants were generated where the aspartate was replaced with serine and cysteine. The D103C and D103S mutants bind the same amount of Zn(II) as wild-type CcrA and exhibited a 10(2)-fold and 10(5)-fold decrease in activity, respectively. Results from solvent isotope, proton inventory, and rapid-scanning visible studies suggest that Asp103 plays a role in generating the enzyme intermediate but does not donate a proton to the enzyme intermediate during the rate-limiting step of the catalytic mechanism.     

Molecular dynamics simulations of class C beta-lactamase from Citrobacter freundii: insights into the base catalyst for acylation

Herein, we present results from molecular dynamics (MD) simulations of the class C beta-lactamase from Citrobacter freundii and its Michaelis complex with aztreonam. Four different configurations of the active site were modeled in aqueous solution, and their relative stability was estimated by means of quantum mechanical energy calculations. For the free enzyme, the energetically most stable configurations present a neutral Lys67 residue or an anionic Tyr150 side chain. Our calculations predict that these two configurations are quite close in terms of free energy, the anionic Tyr150 state being favored by approximately 1 kcal/mol. In contrast, for the noncovalent complex formed between the C. freundii enzyme and aztreonam, the energetic analyses predict that the configuration with the neutral Lys67 residue is much more stable than the anionic Tyr150 one (approximately 20 kcal/mol). Moreover, the MD simulations reveal that the neutral Lys67 state results in a proper enzyme-aztreonam orientation for nucleophilic attack and in a very stable contact between the nucleophilic hydroxyl group of Ser64 and the neutral amino side chain of Lys67. Thus, both the computed free energies and the structural analyses support the assignation of Lys67 as the base catalyst for the acylation step in the native form of the C. freundii enzyme.     

Natures of benzene-water and pyrrole-water interactions in the forms of σ and π types: theoretical studies from clusters to liquid mixture

A combined and sequential use of quantum mechanical (QM) calculations and classical molecular dynamics (MD) simulations was made to investigate the σ and π types of hydrogen bond (HB) in benzene-water and pyrrole-water as clusters and as their liquid mixture, respectively. This paper aims at analyzing similarities and differences of these HBs resulted from QM and MD on an equal footing. Based on the optimized geometry at ωb97xD/aug-cc-pVTZ level of theory, the nature and property of σ and π types of HBs are unveiled by means of atoms in molecules (AIM), natural bond orbital (NBO) and energy decomposition analysis (EDA). In light of the above findings, MD simulation with OPLS-AA and SPC model was applied to study the liquid mixture at different temperatures. The MD results further characterize the behavior and structural properties of σ and π types HBs, which are somewhat different but reasonable for the clusters by QM. Finally, we provide a reasonable explanation for the different solubility between benzene/water and pyrrole/water.

Crystal structures of the cadmium- and mercury-substituted metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require zinc or cadmium for hydrolyzing beta-lactam antibiotics and are inhibited by mercurial compounds. To data, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn(2+)-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam ring. To study the metal coordination further, the crystal structures of a Cd(2+)-bound enzyme and of an Hg(2+)-soaked zinc-containing enzyme have been determined at 2.1 A and 2.7 A, respectively. Given the diffraction resolution, the Cd(2+)-bound enzyme exhibits the same active-site architecture as that of the Zn(2+)-bound enzyme, consistent with the fact that both forms are enzymatically active. The 10-fold reduction in activity of the Cd(2+)-bound molecule compared with the Zn(2+)-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg(2+)-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2-Zn(2+)-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Zn1 and 3.9 A away from the site where Zn2 is located in the 2-Zn(2+)-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.     

The reaction mechanism of paraoxon hydrolysis by phosphotriesterase from combined QM/MM simulations

Molecular dynamics simulations employing combined quantum mechanical and molecular mechanical (QM/MM) potentials have been carried out to investigate the reaction mechanism of the hydrolysis of paraoxon by phosphotriesterase (PTE). We used a dual-level QM/MM approach that synthesizes accurate results from high-level electronic structure calculations with computational efficiency of semiempirical QM/MM potentials for free energy simulations. In particular, the intrinsic (gas-phase) energies of the active site in the QM region are determined by using density functional theory (B3LYP) and second-order M?ller-Plesset perturbation theory (MP2) and the molecular dynamics free energy simulations are performed by using the mixed AM1:CHARMM potential. The simulation results suggest a revised mechanism for the phosphotriester hydrolysis mechanism by PTE. The reaction free energy profile is mirrored by structural motions of the binuclear metal center in the active site. The two zinc ions occupy a compact conformation with an average zinc-zinc distance of 3.5 +/- 0.1 A in the Michaelis complex, whereas it is elongated to 5.3 +/- 0.3 A at the transition state and product state. The substrate is loosely bound to the more exposed zinc ion (Znbeta2+) at an average distance of 3.8 A +/- 0.3 A. The P=O bond of the substrate paraoxon is activated by adopting a tight coordination to the Znbeta2+, releasing the coordinate to the bridging hydroxide ion and increasing its nucleophilicity. It was also found that a water molecule enters into the binding pocket of the loosely bound binuclear center, originally occupied by the nucleophilic hydroxide ion. We suggest that the proton of this water molecule is taken up by His254 at low pH or released to the solvent at high pH, resulting in a hydroxide ion that pulls the Znbeta2+ ion closer to form the compact configuration and restores the resting state of the enzyme.     

Aeromonas proteolytica aminopeptidase: an investigation of the mode of action using a quantum mechanical/molecular mechanical approach

The aminopeptidase of Aeromonas proteolytica (AAP) belongs to the group of metallo-hydrolases that require two divalent cations for full activity. Such binuclear metal centers are found in several aminopeptidases, raising the question whether a common mechanism, at least partly, is likely. We have used a quantum mechanical/molecular mechanical (QM/MM) approach to investigate the reaction mechanism of AAP. Among several possibilities, one reaction path was found to be clearly the most favorable. Beside the chemical transformation steps, effects of the enzyme environment and the influence of the solvent on the catalytic reaction were included in the study. The results are in good agreement with experimental studies and correspond to a high degree to our previous QM/MM calculations on the reaction mechanism of the related binuclear bovine lens leucine aminopeptidase (blLAP), which, although related to the AAP, has different Zn(2+)-coordination spheres and a different catalytic residue. The mechanisms of the two enzymes as suggested in the literature differ on the mode of coordination of the nucleophile and the identity of the general base. However, the results of this and our previous work on blLAP allow us to identify a common mechanism for the two enzymes. This mechanism is probably quite general for binuclear zinc enzymes.     

Crystal structure of the sodium–proton antiporter NhaA dimer and new mechanistic insights

Sodium–proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium–proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium–proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pK_a calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium–proton antiport in NhaA.     

Insights into Cu(I) exchange in HAH1 using quantum mechanical and molecular simulations

The human antioxidant protein, HAH1, is an important participant in a Cu(I) transport chain, delivering one Cu(I) ion to the Wilson's (WND) or Menkes disease protein (MNK). Full geometry optimizations and second-derivative calculations were performed on several binding site models using the B3LYP functional to derive parameters for the construction of a novel molecular mechanical (MM) force field for Cu(I) and its ligating residues in HAH1. MM minimization and molecular dynamics (MD) calculations were then performed using the AMBER suite to validate the newly generated force field. The X-ray crystal structure of the protein and the geometry of the Cu(I) binding site within the protein were reproduced by the MD simulations on the protein based on rmsd and visual inspection, validating the new force field parameters. The results from the quantum mechanical (QM) and MD simulations suggest that either a two- or three-coordinate exchange reaction is preferred and that it is unlikely that a four-coordinate Cu(I) species plays a role in copper exchange.