Sudeep Bhattacharyya

Improved Density Functional Description of the Electrochemistry and Structure−Property Descriptors of Substituted Flavins

The energetics of electrochemical changes have been investigated for several substituted flavins with the M06-L density functional. The reduction potentials for one- and two-electron reductions of these molecules have been determined and the results are consistent with experimental findings with a mean unsigned error of only 42 mV. It is especially noteworthy that the M06-L density functional makes a significant difference in the computed free energy of the first reduction of lumiflavin, which produces a neutral semiquinone. We also investigate the effects of flavin ring substituents on the geometries, charge distributions, reduction potentials, pK(a)s, ionization potentials, electron affinities, hardnesses, softnesses, electrophilic powers, and nucleophilicities.

Evolutionary Basis for the Coupled-domain Motions in Thermus thermophilus Leucyl-tRNA Synthetase

Aminoacyl-tRNA synthetases are multidomain proteins that catalyze the covalent attachment of amino acids to their cognate transfer RNA. Various domains of an aminoacyl-tRNA synthetase perform their specific functions in a highly coordinated manner to maintain high accuracy in protein synthesis in cells. The coordination of their function, therefore, requires communication between domains. In this study we explored the relevance of enzyme motion in domain-domain communications. Specifically, we attempted to probe whether the communication between distantly located domains of a multidomain protein is accomplished through a coordinated movement of structural elements. We investigated the collective motion in Thermus thermophilus leucyl-tRNA synthetase by studying the low frequency normal modes. We identified the mode that best described the experimentally observed conformational changes of T. thermophilus leucyl-tRNA synthetase upon substrate binding and analyzed the correlated and anticorrelated motions between different domains. Furthermore, we used statistical coupling analysis to explore if the amino acid pairs and/or clusters whose motions are thermally coupled have also coevolved. Our study demonstrates that a small number of residues belong to the category whose coupled thermal motions correspond to evolutionary coupling as well. These residue clusters constitute a distinguished set of interacting networks that are sparsely distributed in the domain interface. Residues of these networking clusters are within van der Waals contact, and we suggest that they are critical in the propagation of long range mechanochemical motions in T. thermophilus leucyl-tRNA synthetase.

Critical Role of Substrate Conformational Change in the Proton Transfer Process Catalyzed by 4-Oxalocrotonate Tautomerase

4-Oxalocrotonate tautomerase enzyme (4-OT) catalyzes the isomerization of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate. The chemical process involves two proton transfers, one from a carbon of the substrate to the nitrogen of Pro1 and another from this nitrogen atom to a different carbon of the substrate. In this paper the isomerization has been studied using the combined quantum mechanical and molecular mechanical method with a dual-level treatment of the quantum subsystem employing the MPW1BK density functional as the higher level. Exploration of the potential energy surface shows that the process is stepwise, with a stable intermediate state corresponding to the deprotonated substrate and a protonated proline. The rate constant of the overall process has been evaluated using ensemble-averaged variational transition state theory, including the quantized vibrational motion of a primary zone of active-site atoms and a transmission coefficient based on an ensemble of optimized reaction coordinates to account for recrossing trajectories and optimized multidimensional tunneling. The two proton-transfer steps have similar free energy barriers, but the transition state associated with the first proton transfer is found to be higher in energy. The calculations show that reaction progress is coupled to a conformational change of the substrate, so it is important that the simulation allows this flexibility. The coupled conformational change is promoted by changes in the electron distribution of the substrate that take place as the proton transfers occur.

Role of coupled dynamics in the catalytic activity of prokaryotic-like prolyl-tRNA synthetases.

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNA(Pro) substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNA(Pro) is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In this study, experimental and computational approaches were used to test the hypothesis that deletion of the INS alters the internal protein dynamics leading to reduced catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199-206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Altogether, this study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.

Theoretical Determination of the Redox Potentials of NRH:Quinone Oxidoreductase 2 Using Quantum Mechanical/Molecular Mechanical Simulations

NRH:quinone oxidoreductase 2 (NQO2) is a flavoenzyme that catalyzes a one-step two-electron reduction of quinones. During this enzyme catalysis, the 7,8-dimethyl isoalloxazine (flavin) ring of the enzyme-bound cofactor, flavin adenine dinucleotide (FAD), shuttles between reduced and oxidized states as the enzyme passes through multiple cycles of binding/release of alternate substrates. These redox changes in NQO2, however, lead to unequal charge separation between the flavin ring and the active site, which must be stabilized by reorganization of the surrounding protein matrix. In this study, we have used a combined quantum mechanical/molecular mechanical method to simulate the electron and proton addition reactions of the flavin-bound NQO2. We have computed the redox potentials and pK(a)s of the enzyme-bound flavin. The present work demonstrates that upon reduction, the NQO2 active site stabilizes the flavin anionic hydroquinone state. Simulation data has also allowed quantitative estimation of the electrostatic contributions of active site residues. Their significance in oscillatory redox transition of this flavoenzyme is discussed.

Strictly Conserved Lysine of Prolyl-tRNA Synthetase Editing Domain Facilitates Binding and Positioning of Misacylated tRNA Pro

To ensure high fidelity in translation, many aminoacyl-tRNA synthetases, enzymes responsible for attaching specific amino acids to cognate tRNAs, require proof-reading mechanisms. Most bacterial prolyl-tRNA synthetases (ProRSs) misactivate alanine and employ a post-transfer editing mechanism to hydrolyze Ala-tRNAPro. This reaction occurs in a second catalytic site (INS) that is distinct from the synthetic active site. The 2′-OH of misacylated tRNAPro and several conserved residues in the Escherichia coli ProRS INS domain are directly involved in Ala-tRNAPro deacylation. Although mutation of the strictly conserved lysine 279 (K279) results in nearly complete loss of post-transfer editing activity, this residue does not directly participate in Ala-tRNAPro hydrolysis. We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNAPro and position it in the editing active site. To test this hypothesis, we carried out pKa, charge neutralization, and free-energy of binding calculations. Site-directed mutagenesis and kinetic studies were performed to verify the computational results. The calculations revealed a considerably higher pKa of K279 compared to an isolated lysine and showed that the protonated state of K279 is stabilized by the neighboring acidic residue. However, substitution of this acidic residue with a positively charged residue leads to a significant increase in Ala-tRNAPro hydrolysis, suggesting that enhancement in positive charge density in the vicinity of K279 favors tRNA binding. A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site.

Multiple pathways promote dynamical coupling between catalytic domains in Escherichia coli prolyl-tRNA synthetase.

Aminoacyl-tRNA synthetases are multidomain enzymes that catalyze covalent attachment of amino acids to their cognate tRNA. Cross-talk between functional domains is a prerequisite for this process. In this study, we investigate the molecular mechanism of site-to-site communication in Escherichia coli prolyl-tRNA synthetase (Ec ProRS). Earlier studies have demonstrated that evolutionarily conserved and/or co-evolved residues that are engaged in correlated motion are critical for the propagation of functional conformational changes from one site to another in modular proteins. Here, molecular simulation and bioinformatics-based analysis were performed to identify dynamically coupled and evolutionarily constrained residues that form contiguous pathways of residue-residue interactions between the aminoacylation and editing domains of Ec ProRS. The results of this study suggest that multiple pathways exist between these two domains to maintain the dynamic coupling essential for enzyme function. Moreover, residues in these interaction networks are generally highly conserved. Site-directed changes of on-pathway residues have a significant impact on enzyme function and dynamics, suggesting that any perturbation along these pathways disrupts the native residue-residue interactions that are required for effective communication between the two functional domains. Free energy analysis revealed that communication between residues within a pathway and cross-talk between pathways are important for coordinating functions of different domains of Ec ProRS for efficient catalysis.

Comparison of the Intrinsic Dynamics of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) are an important family of enzymes that catalyze tRNA aminoacylation reaction (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. AARSs are grouped into two broad classes (class I and II) based on sequence/structural homology and mode of their interactions with the tRNA molecule (Ibba and Soll in Annu Rev Biochem 2000, 69:617–650) [1]. As protein dynamics play an important role in enzyme function, we explored the intrinsic dynamics of these enzymes using normal mode analysis and investigated if the two classes and six subclasses (Ia–c and IIa–c) of AARSs exhibit any distinct patterns of motion. The present study found that the intrinsic dynamics-based classification of these enzymes is similar to that obtained based on sequence/structural homology for most enzymes. However, the classification of seryl-tRNA synthetase was not straightforward; the internal mobility patterns of this enzyme are comparable to both IIa and IIb AARSs. This study revealed only a few general mobility patterns in these enzymes—(1) the insertion domain is generally engaged in anticorrelated motion with respect to the catalytic domain for both classes of AARSs and (2) anticodon binding domain dynamics are partly correlated and partly anticorrelated with respect to other domains for class I enzymes. In most of the class II AARSs, the anticodon binding domain is predominately engaged in anticorrelated motion with respect to the catalytic domain and correlated to the insertion domain. This study supports the notion that dynamic-based classification could be useful for functional classification of proteins.

Probing the global and local dynamics of aminoacyl-tRNA synthetases using all-atom and coarse-grained simulations

AbstractCoarse-grained simulations have emerged as invaluable tools for studying conformational changes in biomolecules. To evaluate the effectiveness of computationally inexpensive coarse-grained models in studying global and local dynamics of large protein systems like aminoacyl-tRNA synthetases, we have performed coarse-grained normal mode analysis, as well as principle component analysis on trajectories of all-atom and coarse-grained molecular dynamics simulations for three aminoacyl-tRNA synthetases—Escherichia coli methionyl-tRNA synthetase, Thermus thermophilus leucyl-tRNA synthetase, and Enterococcus faecium prolyl-tRNA synthetase. In the present study, comparison of predicted dynamics based on B-factor and overlap calculations revealed that coarse-grained methods are comparable to the all-atom simulations in depicting the intrinsic global dynamics of the three enzymes. However, the principal component analyses of the motions obtained from the all-atom molecular dynamics simulations provide a superior description of the local fluctuations of these enzymes. In particular, the all-atom model was able to capture the functionally relevant substrate-induced dynamical changes in prolyl-tRNA synthetase. The alteration in the coupled dynamics between the catalytically important proline-binding loop and its neighboring structural elements due to substrate binding has been characterized and reported for the first time. Taken together, the study portrays comparable and contrasting situations in studying the functional dynamics of large multi-domain aminoacyl-tRNA synthetases using coarse-grained and all-atom simulation methods. FigureSubstrate-induced conformational change in E. facium prolyl-tRNA synthetase

Effect of stacking interactions on the thermodynamics and kinetics of lumiflavin: a study with improved density functionals and density functional tight-binding protocol.

The π-π stacking interaction between lumiflavin and a number of π-electron-rich molecules has been studied by density functional theory using several new-generation density functionals. Six known lumiflavin-aromatic adducts were used and the models were evaluated by comparing the geometry and energetics with experimental results. The study found that dispersion-corrected and hybrid functionals with larger (>50%) Hartree-Fock exchanges produced superior results in modeling thermodynamic characteristics of these complexes. The functional producing the best energetics for these model systems was used to study the stacking interactions of lumiflavin with biologically relevant aromatic groups. Additionally, the reduction of flavin-in the presence of both a hydride donor and a nondonor π-electronic system was also studied. Weak interactions were observed in the stacked lumiflavin complexes of benzene, phenol, and indole, mimicking phenyl alanine, tryptophan, and tyrosine side chains, respectively, of an enzyme. The stacked complex of naphthalene and flavin showed little change in flavins redox potential indicating insignificant effect on the thermodynamics of the hydride transfer reaction. In contrast, the hydride transfer reaction with the hydride donor N-methyl nicotinamide tells a different story, as the transition state was found to be strongly impacted by the stacking interactions. A comparison of performance between the density functional theory (DFT) and the computationally less expensive dispersion-corrected self-consistent density functional tight-binding (SCC-DFTB-D) theory revealed that the latter produces consistent energetics for this hydride transfer reaction and additional DFT-computed perturbative corrections could significantly improve these results.