Light-Induced Change of Arginine Conformation Modulates the Rate of Adenosine Triphosphate to Cyclic Adenosine Monophosphate Conversion in the Optogenetic System Containing Photoactivated Adenylyl Cyclase

We report the first computational characterization of an optogenetic system composed of two photosensing BLUF (blue light sensor using flavin adenine dinucleotide) domains and two catalytic adenylyl cyclase (AC) domains. Conversion of adenosine triphosphate (ATP) to the reaction products, cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi), catalyzed by ACs initiated by excitation in photosensing domains has emerged in the focus of modern optogenetic applications because of the request in photoregulated enzymes that modulate cellular concentrations of signaling messengers. The photoactivated AC from the soil bacterium Beggiatoa sp. (bPAC) is an important model showing a considerable increase in the ATP to cAMP conversion rate in the catalytic domain after the illumination of the BLUF domain. The 1 μs classical molecular dynamics simulations reveal that the activation of the BLUF domain leading to tautomerization of Gln49 in the chromophore-binding pocket results in switching of the position of the side chain of Arg278 in the active site of AC. Allosteric signal transmission pathways between Gln49 from BLUF and Arg278 from AC were revealed by the dynamical network analysis. The Gibbs energy profiles of the ATP → cAMP + PPi reaction computed using QM(DFT(ωB97X-D3/6-31G**))/MM(CHARMM) molecular dynamics simulations for both Arg278 conformations in AC clarify the reaction mechanism. In the light-activated system, the corresponding arginine conformation stabilizes the pentacoordinated phosphorus of the α-phosphate group in the transition state, thus lowering the activation energy. Simulations of the bPAC system with the Tyr7Phe replacement in the BLUF demonstrate occurrence of both arginine conformations in an equal ratio, explaining the experimentally observed intermediate catalytic activity of the bPAC-Y7F variant as compared with the dark and light states of the wild-type bPAC

Mechanism of Guanosine Triphosphate Hydrolysis by the Visual Proteins Arl3-RP2: Free Energy Reaction Profiles Computed with Ab Initio Type QM/MM Potentials

We report the results of calculations of the Gibbs energy profiles of the guanosine triphosphate (GTP) hydrolysis by the Arl3-RP2 protein complex using molecular dynamics (MD) simulations with ab initio type QM/MM potentials. The chemical reaction of GTP hydrolysis to guanosine diphosphate (GDP) and inorganic phosphate (Pi) is catalyzed by GTPases, the enzymes, which are responsible for signal transduction in live cells. A small GTPase Arl3, catalyzing the GTP → GDP reaction in complex with the activating protein RP2, constitute an essential part of the human vision cycle. To simulate the reaction mechanism, a model system is constructed by motifs of the crystal structure of the Arl3-RP2 complexed with a substrate analog. After selection of reaction coordinates, energy profiles for elementary steps along the reaction pathway GTP + H2O → GDP + Pi are computed using the umbrella sampling and umbrella integration procedures. QM/MM MD calculations are carried out, interfacing the molecular dynamics program NAMD and the quantum chemistry program TeraChem. Ab initio type QM(DFT)/MM potentials are computed with atom-centered basis sets 6-31G** and two hybrid functionals (PBE0-D3 and ωB97x-D3) of the density functional theory, describing a large QM subsystem. Results of these simulations of the reaction mechanism are compared to those obtained with QM/MM calculations on the potential energy surface using a similar description of the QM part. We find that both approaches, QM/MM and QM/MM MD, support the mechanism of GTP hydrolysis by GTPases, according to which the catalytic glutamine side chain (Gln71, in this system) actively participates in the reaction. Both approaches distinguish two parts of the reaction: the cleavage of the phosphorus-oxygen bond in GTP coupled with the formation of Pi, and the enzyme regeneration. Newly performed QM/MM MD simulations confirmed the profile predicted in the QM/MM minimum energy calculations, called here the pathway-I, and corrected its relief at the first elementary step from the enzyme–substrate complex. The QM/MM MD simulations also revealed another mechanism at the part of enzyme regeneration leading to pathway-II. Pathway-II is more consistent with the experimental kinetic data of the wild-type complex Arl3-RP2, whereas pathway-I explains the role of the mutation Glu138Gly in RP2 slowing down the hydrolysis rate

Dynamical properties of enzyme-substrate complexes disclose substrate specificity of the SARS-CoV-2 main protease as characterized by the electron density descriptors

A dynamical approach is proposed to discriminate between reactive (rES) and nonreactive (nES) enzyme-substrate complexes taking the SARS-CoV-2 main protease (Mpro) as an important example. Molecular dynamics simulations with the quantum mechanics/molecular mechanics potentials (QM(DFT)/MM-MD) followed by the electron density analysis are employed to evaluate geometry and electronic properties of the enzyme with different substrates along MD trajectories. We demonstrate that mapping the Laplacian of the electron density and the electron localization function provides easily visible images of the substrate activation that allow one to distinguish rES and nES. The computed fractions of reactive enzyme-substrate complexes along MD trajectories well correlate with the findings of recent experimental studies on the substrate specificity of Mpro. The results of our simulations demonstrate the role of the theory level used in QM subsystems for a proper description of the nucleophilic attack of the catalytic cysteine residue in Mpro. The activation of the carbonyl group of a substrate is correctly characterized with the hybrid DFT functional PBE0, whereas the use of a GGA-type PBE functional, that lacks the admixture of the Hartree-Fock exchange fails to describe activation

Modeling Spectral Tuning in Red Fluorescent Proteins Using the Dipole Moment Variation upon Excitation

We describe a model for spectral tuning in red fluorescent proteins (RFPs) based on the relation between an electronic structure descriptor, the dipole moment variation upon excitation (DMV), and the excitation energy of a protein. This approach aims to overcome a problem of accurate prediction of excitation energies in RFPs, which span a very narrow window of band maxima. The latter roughly corresponds to an energy gap of 0.1 eV, which is comparable with typical errors in calculations of the excitation energy by conventional quantum chemistry methods. In this work, we demonstrate a strong quantitative correlation between DMV values, obtained computationally with modest efforts, and excitation energies ΔEex at the experimental excitation band maxima for a series of RFPs with the bands between 570 and 605 nm. Protein models are constructed by motifs of the relevant crystal structures, and atomic coordinates are optimized in quantum mechanics/molecular mechanics (QM/MM) calculations with QM-subsystems composed of large chromophore-containing regions. DMV values are evaluated with the electron density computed at the TDDFT level using several functionals and basis sets. We demonstrate that the results obtained with the CAM-B3LYP, BHHLYP and M06-2X functionals demonstrate favorable correlations between DMV and ΔEex with the mean absolute error less than 0.01 eV. Taking into account the solid theoretical grounds of the relation between the DMV and the excitation energy in fluorescent proteins, the described modeling strategy presents a rational tool for spectral tuning in these efficient markers for in vivo imaging

Evolution of Ceftriaxone Resistance of Penicillin-Binding Proteins 2 Revealed by Molecular Modeling

Penicillin-binding proteins 2 (PBP2) are critically important enzymes in the formation of the bacterial cell wall. Inhibition of PBP2 is utilized in the treatment of various diseases, including gonorrhea. Ceftriaxone is the only drug used to treat gonorrhea currently, and recent growth in PBP2 resistance to this antibiotic is a serious threat to human health. Our study reveals mechanistic aspects of the inhibition reaction of PBP2 from the wild-type FA19 strain and mutant 35/02 and H041 strains of Neisseria Gonorrhoeae by ceftriaxone. QM(PBE0-D3/6-31G**)/MM MD simulations show that the reaction mechanism for the wild-type PBP2 consists of three elementary steps including nucleophilic attack, C–N bond cleavage in the β-lactam ring and elimination of the leaving group in ceftriaxone. In PBP2 from the mutant strains, the second and third steps occur simultaneously. For all considered systems, the acylation rate is determined by the energy barrier of the first step that increases in the order of PBP2 from FA19, 35/02 and H041 strains. Dynamic behavior of ES complexes is analyzed using geometry and electron density features including Fukui electrophilicity index and Laplacian of electron density maps. It reveals that more efficient activation of the carbonyl group of the antibiotic leads to the lower energy barrier of nucleophilic attack and larger stabilization of the first reaction intermediate. Dynamical network analysis of MD trajectories explains the differences in ceftriaxone binding affinity: in PBP2 from the wild-type strain, the β3-β4 loop conformation facilitates substrate binding, whereas in PBP2 from the mutant strains, it exists in the conformation that is unfavorable for complex formation. Thus, we clarify that the experimentally observed decrease in the second-order rate constant of acylation (k2/KS) in PBP2 from the mutant strains is due to both a decrease in the acylation rate constant k2 and an increase in the dissociation constant KS

Influence of the Active Site Flexibility on the Efficiency of Substrate Activation in the Active Sites of Bi-Zinc Metallo-β-Lactamases

The influence of the active site flexibility on the efficiency of catalytic reaction is studied by taking two members of metallo-β-lactamases, L1 and NDM-1, with the same substrate, imipenem. Active sites of these proteins are covered by L10 loops, and differences in their amino acid compositions affect their rigidity. A more flexible loop in the NDM-1 brings additional flexibility to the active site in the ES complex. This is pronounced in wider distributions of key interatomic distances, such as the distance of the nucleophilic attack, coordination bond lengths, and covalent bond lengths in the substrate. Substrate activation, quantified by Fukui electrophilicity index of the carbonyl carbon atom of the substrate, is also sensitive to the active site flexibility. In the tighter and more rigid L1 enzyme-substrate complex, the substrate is activated more efficiently. In the NDM-1 containing system, only one third of the states are activated to the same extent. Other fractions demonstrate lower substrate activation. Efficiency of the substrate activation and rigidity of the ES complex influence the following chemical reaction. In the more rigid L1-containing system, the reaction barrier of the first step of the reaction is lower, and the first intermediate is more stabilized compared to the NDM-1 containing system

Evolution of Ceftriaxone Resistance of Penicillin-Binding Proteins 2 Revealed by Molecular Modeling

Penicillin-binding proteins 2 (PBP2) are critically important enzymes in the formation of the bacterial cell wall. Inhibition of PBP2 is utilized in the treatment of various diseases, including gonorrhea. Ceftriaxone is the only drug used to treat gonorrhea currently, and recent growth in PBP2 resistance to this antibiotic is a serious threat to human health. Our study reveals mechanistic aspects of the inhibition reaction of PBP2 from the wild-type FA19 strain and mutant 35/02 and H041 strains of Neisseria Gonorrhoeae by ceftriaxone. QM(PBE0-D3/6-31G**)/MM MD simulations show that the reaction mechanism for the wild-type PBP2 consists of three elementary steps including nucleophilic attack, C–N bond cleavage in the β-lactam ring and elimination of the leaving group in ceftriaxone. In PBP2 from the mutant strains, the second and third steps occur simultaneously. For all considered systems, the acylation rate is determined by the energy barrier of the first step that increases in the order of PBP2 from FA19, 35/02 and H041 strains. Dynamic behavior of ES complexes is analyzed using geometry and electron density features including Fukui electrophilicity index and Laplacian of electron density maps. It reveals that more efficient activation of the carbonyl group of the antibiotic leads to the lower energy barrier of nucleophilic attack and larger stabilization of the first reaction intermediate. Dynamical network analysis of MD trajectories explains the differences in ceftriaxone binding affinity: in PBP2 from the wild-type strain, the β3-β4 loop conformation facilitates substrate binding, whereas in PBP2 from the mutant strains, it exists in the conformation that is unfavorable for complex formation. Thus, we clarify that the experimentally observed decrease in the second-order rate constant of acylation (k2/KS) in PBP2 from the mutant strains is due to both a decrease in the acylation rate constant k2 and an increase in the dissociation constant KS

On the Fly Determination of the Substrate Activation in Hydrolases Using a Neural Network

Active sites of enzymes are able to activate substrates and perform chemical reactions that cannot occur in solutions. We focus here on hydrolysis reactions catalyzed by enzymes and initiated by the nucleophilic attack of the carbonyl carbon atom of the substrate. From the electronic structure standpoint, substrate activation can be characterized in terms of the Laplacian of the electron density. This is a simple and easily visible imaging that allows one to “visualize” electrophilic site on the carbonyl carbon atom, that is presented only in the activated species. The efficiency of the substrate activation by the enzyme can be quantified from the ratio of reactive and nonreactive states from the QM/MM MD trajectories. We propose a neural network that assigns the Laplacian of electron density maps to the reactive and nonreactive species. The neural network is trained on the cysteine protease enzyme-substrate complexes and successfully validated on the zinc-containing hydrolase, thus showing a wide range of applications of the proposed approach

Molecular Mechanism of Light-Induced Acceleration of the Adenosine Triphosphate Conversion to the Cyclic Adenosine Monophosphate Catalyzed by the Photoactivated Adenylyl Cyclase bPAC

We report the first computational characterization of an optogenetic system composed of two photosensing BLUF (blue light sensor using flavin adenine dinucleotide) domains and two catalytic adenylyl cyclase (AC) domains. Conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi) catalyzed by ACs coupled with excitation in photosensing domains has emerged in the focus of modern optogenetic applications because of the request in photoregulated enzymes to modulate cellular concentrations of signaling messengers. The photoactivated adenylyl cyclase from the soil bacterium Beggiatoa sp. (bPAC) is an important model showing considerable increase of the ATP to cAMP conversion rate in the catalytic domain after the illumination of the BLUF domain. The 1 μs classical molecular dynamics simulations reveal that the activation of the BLUF domain leading to tautomerization of Gln49 in the chromophore binding pocket results in switching of position of the side chain of Arg278 in the active site of AC. Allosteric signal transmission pathways between Gln49 from BLUF and Arg278 from AC were revealed by the dynamical network analysis. The Gibbs energy profiles of the ATP → cAMP + PPi reaction computed using QM(DFT(ωB97X-D3/6-31G**))/MM(CHARMM) molecular dynamics simulations for both Arg278 conformations in AC clarify the reaction mechanism. In the light-activated system, the corresponding arginine conformation stabilizes the pentacoordinated phosphorus of the α-phosphate group in the transition state, thus lowering the activation energy. Simulations of the bPAC system with the Tyr7Phe replacement in BLUF demonstrate occurrence of both arginine conformations in an equal ratio, explaining the experimentally observed intermediate catalytic activity of the bPAC-Y7F variant as compared with the dark and light states of the wild type bPAC. </p

Molecular basis of the substrate specificity of the Pd-PTE phosphotriesterase: a combined QM/MM MD and electron density study

The increase of organophosphorus compounds, pesticides and flame-retardants, in wastes is an emerging ecological problem. Bacterial phosphotriesterases are capable for hydrolysis of some of them. We utilize modern molecular modeling tools to study hydrolysis mechanism of organophosphorus compounds with different leaving groups by phosphotriesterase from Pseudomonas diminuta (Pd-PTE). We compute Gibbs energy profiles for enzymes with different cations in the active site: native Zn2+ cations; Co2+ cations that increase the steady-state rate constant. A high-spin state of the cobalt-containing active site catalyzes hydrolysis. Reaction happens with two elementary steps via formation of the pentacoordinated intermediate. For substrates with good leaving groups the reaction proceeds with low energy barriers with both Zn2+ and Co2+ cations in the active site, thus the product release is likely to be a limiting step. For substrates with poor leaving groups, reaction products are destabilized relative to the ES complex that suppresses the reaction. Electron density and geometry analysis of the QM/MM MD trajectories of the intermediate states with all considered compounds allow us to discriminate substrates by their ability to be hydrolyzed by the Pd-PTE. These criteria is can be utilized to predict whether novel organophosphorus compounds can be hydrolyzed by the Pd-PTE