Ab initio molecular dynamics-based assignment of the protonation state of pepstatin A/HIV-1 protease cleavage site

A recent 13C NMR experiment (Smith et al. Nature Struct. Biol. 1996, 3, 946-950) on the Asp 25-Asp25' dyad in pepstatin A/HIV-1 protease measured two separate resonance lines, which were interpreted as being a singly protonated dyad. We address this issue by performing ab initio molecular dynamics calculations on models for this site accompanied by calculations of 13C NMR chemical shifts and isotopic shifts. We find that already on the picosecond time-scale the model proposed by Smith et al. is not stable and evolves toward a different monoprotonated form whose NMR pattern differs from the experimental one. We suggest, instead, a different protonation state in which both aspartic groups are protonated. Despite the symmetric protonation state, the calculated 13C NMR properties are in good agreement with the experiment. We rationalize this result using a simple valence bond model, which explains the chemical inequality of the two C sites. The model calculations, together with our calculations on the complex, allow also the rationalization of 13C NMR properties on other HIV-1 PR/inhibitor complexes. Both putative binding of the substrate to the free enzyme, which has the dyad singly protonated (Piana, S.; Carloni, P. Proteins: Struct., Funct., Genet. 2000, 39, 26-36), and pepstatin A binding to the diprotonated form are consistent with the inverse solvent isotope effect on the onset of inhibition of pepsin by pepstatin and the kinetic iso-mechanism proposed for aspartic proteases (Cho, T.-K.; Rebholz, K.; Northrop, D.B. Biochemistry 1994, 33, 9637-9642).     

Theoretical investigations of structure and enzymatic mechanisms of aspartyl proteinases : Part I. Ab-initio calculations on an active site model: hydrogen diformiate with H2O and H3O

The active site of aspartyl proteinases (Asp) was modelled as two formiates connected with a proton and set in geometry corresponding to Asp 32 and Asp 215 side chain carboxylate groups of endothiapepsin. The shared solvent molecule was alternatively H₂O and H₃O⁺. Their positions and those of hydrogen-bonded protons were optimized using the STO-3G basis set. Full geometry optimizations were made of the hydrogen diformiate complexes with H₂O and H₃O⁺. Asymmetric hydrogen-bonded structures resulted from these calculations, except for the fully optimized complex with H₂O. In the complexes with H₃O⁺, one proton moved consistently to the proximate carboxylic oxygen yielding a neutral, hydrated formic acid dimer. Interaction energies and proton potential energy curves were calculated using the 4-31G basis set. The interaction energy with H₂O was found to be 20.49 kcal mol⁻¹ and 202.75 kcal mol⁻¹ with H₃O⁺.     

Deciphering the catalytic amino acid residues of l-2-haloacid dehalogenase (DehL) from Rhizobium sp. RC1: An in silico analysis

The l-2-haloacid dehalogenases (EC 3.8.1.2) specifically cleave carbon-halogen bonds in the L-isomers of halogenated organic acids. These enzymes have potential applications for the bioremediation and synthesis of various industrial products. One such enzyme is DehL, the l-2-haloacid dehalogenase from Rhizobium sp. RC1, which converts the L-isomers of 2-halocarboxylic acids into the corresponding D-hydroxycarboxylic acids. However, its catalytic mechanism has not been delineated, and to enhance its efficiency and utility for environmental and industrial applications, knowledge of its catalytic mechanism, which includes identification of its catalytic residues, is required. Using ab initio fragment molecular orbital calculations, molecular mechanics Poisson-Boltzmann surface area calculations, and classical molecular dynamic simulation of a three-dimensional model of DehL-l-2-chloropropionic acid complex, we predicted the catalytic residues of DehL and propose its catalytic mechanism. We found that when Asp13, Thr17, Met48, Arg51, and His184 were individually replaced with an alanine in silico, a significant decrease in the free energy of binding for the DehL-l-2-chloropropionic acid model complex was seen, indicating the involvement of these residues in catalysis and/or structural integrity of the active site. Furthermore, strong inter-fragment interaction energies calculated for Asp13 and L-2-chloropropionic acid, and for a water molecule and His184, and maintenance of the distances between atoms in the aforementioned pairs during the molecular dynamics run suggest that Asp13 acts as the nucleophile and His184 activates the water involved in DehL catalysis. The results of this study should be important for the rational design of a DehL mutant with improved catalytic efficiency.     

Ionization state of the catalytic dyad Asp25/25' in the HIV-1 protease: NMR studies of site-specifically 13C labelled HIV-1 protease prepared by total chemical synthesis

Total chemical synthesis was used to site-specifically (13)C-label active site Asp25 and Asp25' residues in HIV-1 protease and in several chemically synthesized analogues of the enzyme molecule. (13)C NMR measurements were consistent with a monoprotonated state for the catalytic dyad formed by the interacting Asp25, Asp25' side chain carboxyls.     

Differential effects of the Zn-His-Bkb vs Zn-His-[Asp/Glu] triad on Zn-core stability and reactivity

The most common partner of the Zn-bound His is the Asp/Glu carboxylate side chain in catalytic Zn sites and the backbone (Bkb) carbonyl group in structural Zn sites. To elucidate the factors governing the selection of the second-shell partner of the Zn-bound His in structural/catalytic Zn sites, systematic studies using density functional theory and continuum dielectric calculations were performed to determine the relative contributions of the second-shell Bkb carbonyl and the Asp/Glu carboxylate to the Zn-core stability and reactivity. The results show that the contributions of the second-shell Bkb carbonyl and Asp/Glu carboxylate to the Zn-core stability depend mainly on the solvent accessibility of the Zn-site and the composition of the Zn-core. They reveal the advantage of a second-shell Bkb carbonyl in anionic Zn cavities: it stabilizes anionic, buried Zn-cores more than the corresponding negatively charged Asp/Glu carboxylate, thus explaining the absence of the Zn-His-Asp/Glu triad in structural [Zn(Cys)3(His)]- cores. They also reveal the advantage of a second-shell Asp/Glu carboxylate in catalytic Zn-cores: relative to a Bkb carbonyl group, it increases (i) the HOMO energy of the cationic/neutral zinc core, (ii) the reactivity of the attacking Zn-bound OH-, (iii) electron transfer to the substrate, and (iv) the stability of the metal complex upon electron transfer. Furthermore, a second-shell Asp/Glu carboxylate could facilitate product release in the common cationic catalytic cores, by acting as a proton acceptor of the Zn-bound His creating an Asp...His- dyad that stabilizes the zinc dication more than the respective Bkb...His0 dyad.     

Long‐Range Electrostatics‐Induced Two‐Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site

Neutron crystallography was used to directly locate two protons before and after a pH‐induced two‐proton transfer between catalytic aspartic acid residues and the hydroxy group of the bound clinical drug darunavir, located in the catalytic site of enzyme HIV‐1 protease. The two‐proton transfer is triggered by electrostatic effects arising from protonation state changes of surface residues far from the active site. The mechanism and pH effect are supported by quantum mechanics/molecular mechanics (QM/MM) calculations. The low‐pH proton configuration in the catalytic site is deemed critical for the catalytic action of this enzyme and may apply more generally to other aspartic proteases. Neutrons therefore represent a superb probe to obtain structural details for proton transfer reactions in biological systems at a truly atomic level.     

Methodological aspects of QM/MM calculations: A case study on matrix metalloproteinase‐2

We address methodological issues in quantum mechanics/molecular mechanics (QM/MM) calculations on a zinc‐dependent enzyme. We focus on the first stage of peptide bond cleavage by matrix metalloproteinase‐2 (MMP‐2), that is, the nucleophilic attack of the zinc‐coordinating water molecule on the carbonyl carbon atom of the scissile fragment of the substrate. This step is accompanied by significant charge redistribution around the zinc cation, bond cleavage, and bond formation. We vary the size and initial geometry of the model system as well as the computational protocol to demonstrate the influence of these choices on the results obtained. We present QM/MM potential energy profiles for a set of snapshots randomly selected from QM/MM‐based molecular dynamics simulations and analyze the differences in the computed profiles in structural terms. Since the substrate in MMP‐2 is located on the protein surface, we investigate the influence of the thickness of the water layer around the enzyme on the QM/MM energy profile. Thin water layers (0–2 Å) give unrealistic results because of structural reorganizations in the active‐site region at the protein surface. A 12 Å water layer appears to be sufficient to capture the effect of the solvent; the corresponding QM/MM energy profile is very close to that obtained from QM/MM/SMBP calculations using the solvent macromolecular boundary potential (SMBP). We apply the optimized computational protocol to explain the origin of the different catalytic activity of the Glu116Asp mutant: the energy barrier for the first step is higher, which is rationalized on structural grounds. © 2016 Wiley Periodicals, Inc.     

How does the cAMP-dependent protein kinase catalyze the phosphorylation reaction: an ab initio QM/MM study

We have carried out density functional theory QM/MM calculations on the catalytic subunit of cAMP-dependent protein kinase (PKA). The QM/MM calculations indicate that the phosphorylation reaction catalyzed by PKA is mainly dissociative, and Asp166 serves as the catalytic base to accept the proton delivered by the substrate peptide. Among the key interactions in the active site, the Mg(2+) ions, glycine rich loop, and Lys72 are found to stabilize the transition state through electrostatic interactions. On the other hand, Lys168, Asn171, Asp184, and the conserved waters bound to Mg(2+) ions do not directly contribute to lower the energy barrier of the phosphorylation reaction, and possible roles for these residues are proposed. The QM/MM calculations with different QM/MM partition schemes or different initial structures yield consistent results. In addition, we have carried out 12 ns molecular dynamics simulations on both wild type and K168A mutated PKA, respectively, to demonstrate that the catalytic role of Lys168 is to keep ATP and substrate peptide in the near-attack reactive conformation.     

A density functional study of the hydrogen-bond network within the HIV-1 protease catalytic site cleft

The relative energy between two different protonation sites of the Asp25' catalytic site residue is computed and analyzed for various HIV-1 Protease/inhibitor complexes and compared to the wild-type structure. By comparing calculations of negatively charged fragments of gradually increasing size up to 105 atoms we show that correct modeling of the HIV-1 Protease active site requires much larger models than the commonly used acetic acid/acetate moieties. The energy difference between the two proposed protonation sites decreases as the size of the system increases and tends to converge only when the entire catalytic triad of both monomers is taken into account. The importance of the Gly27 backbone amine groups in the stabilization of the negative charge within the catalytic site cleft is revealed. Comparison of the wild-type structure with the structures from various Pr/drug complexes indicates that the HIV-1 protease has a particular catalytic site flexibility.     

Analysis of classical and quantum paths for deprotonation of methylamine by methylamine dehydrogenase.

The hydrogen-transfer reaction catalysed by methylamine dehydrogenase (MADH) with methylamine (MA) as substrate is a good model system for studies of proton tunnelling in enzyme reactions--an area of great current interest--for which atomistic simulations will be vital. Here, we present a detailed analysis of the key deprotonation step of the MADH/MA reaction and compare the results with experimental observations. Moreover, we compare this reaction with the related aromatic amine dehydrogenase (AADH) reaction with tryptamine, recently studied by us, and identify possible causes for the differences observed in the measured kinetic isotope effects (KIEs) of the two systems. We have used combined quantum mechanics/molecular mechanics (QM/MM) techniques in molecular dynamics simulations and variational transition state theory with multidimensional tunnelling calculations averaged over an ensemble of paths. The results reveal important mechanistic complexity. We calculate activation barriers and KIEs for the two possible proton transfers identified-to either of the carboxylate oxygen atoms of the catalytic base (Asp428beta)-and analyse the contributions of quantum effects. The activation barriers and tunnelling contributions for the two possible proton transfers are similar and lead to a phenomenological activation free energy of 16.5+/-0.9 kcal mol(-1) for transfer to either oxygen (PM3-CHARMM calculations applying PM3-SRP specific reaction parameters), in good agreement with the experimental value of 14.4 kcal mol(-1). In contrast, for the AADH system, transfer to the equivalent OD1 was found to be preferred. The structures of the enzyme complexes during reaction are analysed in detail. The hydrogen bond of Thr474beta(MADH)/Thr172beta(AADH) to the catalytic carboxylate group and the nonconserved active site residue Tyr471beta(MADH)/Phe169beta(AADH) are identified as important factors in determining the preferred oxygen acceptor. The protein environment has a significant effect on the reaction energetics and hence on tunnelling contributions and KIEs. These environmental effects, and the related clearly different preferences for the two carboxylate oxygen atoms (with different KIEs) in MADH/MA and AADH/tryptamine, are possible causes of the differences observed in the KIEs between these two important enzyme reactions.     

QM/MM modelling of oleamide hydrolysis in fatty acid amide hydrolase (FAAH) reveals a new mechanism of nucleophile activation

Fatty acid amide hydrolase (FAAH), a promising target for the treatment of several central and peripheral nervous system disorders, such as anxiety, pain and hypertension, has an unusual catalytic site, and its mechanism has been uncertain; hybrid quantum mechanics/molecular mechanics (QM/MM) calculations reveal a new mechanism of nucleophile activation (involving a Lys-Ser-Ser catalytic triad), with potentially crucial insights for the design of potent and selective inhibitors.     

Stepwise Versus Concerted Mechanisms in General‐Base Catalysis by Serine Proteases

General‐base catalysis in serine proteases still poses mechanistic challenges despite decades of research. Whether proton transfer from the catalytic Ser to His and nucleophilic attack on the substrate are concerted or stepwise is still under debate, even for the classical Asp‐His‐Ser catalytic triad. To address these key catalytic steps, the transformation of the Michaelis complex to tetrahedral complex in the covalent inhibition of two prototype serine proteases was studied: chymotrypsin (with the catalytic triad) inhibition by a peptidyl trifluoromethane and GlpG rhomboid (with Ser‐His dyad) inhibition by an isocoumarin derivative. The sampled MD trajectories of averaged pK_a values of catalytic residues were QM calculated by the MD‐QM/SCRF(VS) method on molecular clusters simulating the active site. Differences between concerted and stepwise mechanisms are controlled by the dynamically changing pK_a values of the catalytic residues as a function of their progressively reduced water exposure, caused by the incoming ligand.     

Protonation states of the catalytic dyad of β-secretase (BACE1) in the presence of chemically diverse inhibitors: a molecular docking study

In this molecular docking study, the protonation states of the catalytic Asp dyad of the beta-secretase (BACE1) enzyme in the presence of eight chemically diverse inhibitors have been predicted. BACE1 catalyzes the rate-determining step in the generation of Alzheimer amyloid beta peptides and is widely considered as a promising therapeutic target. All the inhibitors were redocked into their corresponding X-ray structures using a combination of eight different protonation states of the Asp dyad for each inhibitor. Five inhibitors were primarily found to favor two different monoprotonated states, and the remaining three favor a dideprotonated state. In addition, five of them exhibited secondary preference for a diprotonated state. These results show that the knowledge of a single protonation state of the Asp dyad is not sufficient to search for the novel inhibitors of BACE1 and the most plausible state for each inhibitor must be determined prior to conducting in-silico screening.     

Common system setup for the entire catalytic cycle of cytochrome P450(cam) in quantum mechanical/molecular mechanical studies

We describe a system setup that is applicable to all species in the catalytic cycle of cytochrome P450(cam). The chosen procedure starts from the X-ray coordinates of the ferrous dioxygen complex and follows a protocol that includes the careful assignment of protonation states, comparison between different conceivable hydration schemes, and system preparation through a series of classical minimizations and molecular dynamics (MD) simulations. The resulting setup was validated by quantum mechanical/molecular mechanical (QM/MM) calculations on the resting state, the pentacoordinated ferric and ferrous complexes, Compound I, the transition state and hydroxo intermediate of the C--H hydroxylation reaction, and the product complex. The present QM/MM results are generally consistent with those obtained previously with individual setups. Concerning hydration, we find that saturating the protein interior with water is detrimental and leads to higher structural flexibility and catalytically inefficient active-site geometries. The MD simulations favor a low water density around Asp251 that facilitates side chain rotation of protonated Asp251 during the conversion of Compound 0 to Compound I. The QM/MM results for the two preferred hydration schemes (labeled SE-1 and SE-4) are similar, indicating that slight differences in the solvation close to the active site are not critical as long as camphor and the crystallographic water molecules preserve their positions in the experimental X-ray structures.     

Water-assisted reaction mechanism of monozinc beta-lactamases

Hybrid Car-Parrinello QM/MM calculations are used to investigate the reaction mechanism of hydrolysis of a common beta-lactam substrate (cefotaxime) by the monozinc beta-lactamase from Bacillus cereus (BcII). The calculations suggest a fundamental role for an active site water in the catalytic mechanism. This water molecule binds the zinc ion in the first step of the reaction, expanding the zinc coordination number and providing a proton donor adequately oriented for the second step. The free energy barriers of the two reaction steps are similar and consistent with the available experimental data. The conserved hydrogen bond network in the active site, defined by Asp120, Cys221, and His263, not only contributes to orient the nucleophile (as already proposed), but it also guides the second catalytic water molecule to the zinc ion after the substrate is bound. The hydrolysis reaction in water has a relatively high free energy barrier, which is consistent with the stability of cefotaxime in water solution. The modeled Michaelis complexes for other substrates are also characterized by the presence of an ordered water molecule in the same position, suggesting that this mechanism might be general for the hydrolysis of different beta-lactam substrates.     

Molecular simulation studies to reveal the binding mechanisms of shikonin derivatives inhibiting VEGFR-2 kinase

Traditional vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitors can manage angiogenesis; however, severe toxicity and resistance limit their long-term applications in clinical therapy. Shikonin (SHK) and its derivatives could be promising to inhibit the VEGFR-2 mediated angiogenesis, as they are reported to bind in the catalytic kinase domain with low affinity. However, the detailed molecular insights and binding dynamics of these natural inhibitors are unknown, which is crucial for potential SHK based lead design. Therefore, the present study employed molecular modeling and simulations techniques to get insight into the binding behaviors of SHK and its two derivates, β-hydroxyisovalerylshikonin (β-HIVS) and acetylshikonin (ACS). Here the intermolecular interactions between protein and ligands were studied by induced fit docking approach, which were further evaluated by treating QM/MM (quantum mechanics/molecular mechanics) and molecular dynamics (MD) simulation. The result showed that the naphthazarin ring of the SHK derivates is vital for strong binding to the catalytic domain; however, the binding stability can be modulated by the side chain modification. Because of having electrostatic potential, this ring makes essential interactions with the DFG (Asp1046 and Phe1047) motif and also allows interacting with the allosteric binding site. Taken together, the studies will advance our knowledge and scope for the development of new selective VEGFR-2 inhibitors based on SHK and its analogs.     

Factors governing the protonation state of Zn-bound histidine in proteins: a DFT/CDM study

We have performed systematic theoretical studies to elucidate the factors governing the His protonation/deprotonation state in Zn-binding sites, especially those containing the ubiquitous Zn-His-Asp/Glu triad. Specifically, we have addressed the following three questions: (1) How does the transfer of the Zn-bound His imidazole proton to the second-shell Asp/Glu carboxylate oxygen depend on the composition of the other first-shell ligands and the solvent accessibility of the metal-binding site? (2) Can any second-shell ligand with a proton acceptor group such as the backbone carbonyl oxygen also act as a proton acceptor? (3) What is the effect of the Asp/Glu in the Zn-His-Asp/Glu triad on the Zn-bound water protonation state? To address these questions, we used a combination of quantum mechanical and continuum dielectric methods to compute the free energies for deprotonating a Zn-bound imidazole/water in various Zn complexes. The calculations show that whether the Zn-bound His is protonated or deprotonated depends on (1) the solvent accessibility of the metal-binding site, and (2) the Lewis acid ability of Zn, which is indirectly determined by both the first- and the second-shell Zn ligands. The calculations also show that the effect of the Zn-His-Asp/Glu interaction on the nucleophilicity of the Zn-bound water depends on the solvent accessibility of the catalytic Zn site. Furthermore, they show that the Asp/Glu side chain in the Zn-His-Asp/Glu triad can increase the negative charge of its partner, His, and create an anionic hole that may stabilize a cation in buried cavities, provided that the Zn complex is cationic/neutral. The findings of this work are in accord with available experimental data.     

Binding free energy calculations to rationalize the interactions of huprines with acetylcholinesterase

In the present study, the binding free energy of a family of huprines with acetylcholinesterase (AChE) is calculated by means of the free energy perturbation method, based on hybrid quantum mechanics and molecular mechanics potentials. Binding free energy calculations and the analysis of the geometrical parameters highlight the importance of the stereochemistry of huprines in AChE inhibition. Binding isotope effects are calculated to unravel the interactions between ligands and the gorge of AChE. New chemical insights are provided to explain and rationalize the experimental results. A good correlation with the experimental data is found for a family of inhibitors with moderate differences in the enzyme affinity. The analysis of the geometrical parameters and interaction energy per residue reveals that Asp72, Glu199, and His440 contribute significantly to the network of interactions between active site residues, which stabilize the inhibitors in the gorge. It seems that a cooperative effect of the residues of the gorge determines the affinity of the enzyme for these inhibitors, where Asp72, Glu199, and His440 make a prominent contribution.     

A Novel Bicyclophosphite Ligand Directly Yields Rhodium Hydride Complexes

 Complexation of Rh(I) with o, o′-dimethylene-(tris-p-cresyl)-bicyclophosphite (BCP, 1) has been investigated in solution by NMR, semi-empirical quantum mechanical, and molecular mechanics calculations. ¹H and ³¹P NMR spectroscopic data show that when the BCP/Rh ratio exceeds 2, Rh hydride complexes of the composition RhH(BCP)₃ and RhH(BCP)₄ are formed. The source of the hydride ion is the ligand itself; most probably, H⁻ originates from the bridging CH₂ groups of BCP. The chemical shifts of these protons are sensitive to complexation due to the considerable electron density of HOMO and LUMO at one of the bridging CH₂ moieties. Molecular mechanics simulations of the molecular structure of these complexes show that two cavities are formed in [Rh(BCP)₃]⁺ by the aromatic rings of the ligands. These cavities may alternatively open and close, thus providing for a flexibly shielded catalytic site which explains the unusual catalytic behaviour of Rh complexes with BCP in hydrogenation and hydroformylation reactions.