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DNA polymerase β catalytic efficiency mirrors the Asn279–dCTP H-bonding strength
Abstract
Ternary complexes of wild type or mutant form of human DNA polymerase beta (pol beta) bound to DNA and dCTP substrates were studied by molecular dynamics (MD) simulations. The occurrences of contact configurations (CC) of structurally important atom pairs were sampled along the MD trajectories, and converted into free-energy differences, DeltaG(CC). DeltaG(CC) values were correlated with the experimental binding and catalytic free energies for the wild type pol beta and its Arg183Ala, Tyr271Ala, Asp276Val, Lys280Gly, Arg283Ala, and Glu295Ala mutants. The correlation coefficients show that the strength of the H-bond between dCTP and Asn279 is a strong predictor of the mutation-induced changes in the catalytic efficiency of pol beta. This finding is consistent with the view that enzyme preorganization plays a major role in controlling DNA polymerase specific activity.
... Endonucleases require one, two or three divalent metal ions, such as Mn 2+ or Mg 2+ in the catalytic site [7][8][9][10][11][12][13]. It is believed that in thermal environment, such as the one Thermotoga maritima lives in at temperatures around 80 °C, the most suitable metal ion for this kind of system is Mg 2+ [14]. ...
... Usually such a structure would suggest a transition state in the reaction pathway but the Mg 2+ ions effectively stabilize the energy of pentacovalent intermediate to make it a stable minimum. In order to check this structure and its energy we performed (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) represent steps on the reaction path, in which 1 is reactant and 16 is product. Values for distances between atoms Cyt6 03′-P (3DR7) (green) and water 1863 OH2-P (3DR7) (blue) are marked on the y2 axis a separate calculation. ...
A protein, Tm1631 from the hyperthermophilic organism Thermotoga maritima belongs to a domain of unknown function protein family. It was predicted that Tm1631 binds with the DNA and that the Tm1631-DNA complex is an endonuclease repair system with a DNA repair function (Konc et al. PLoS Comput Biol 9(11): e1003341, 2013). We observed that the severely bent, strained DNA binds to the protein for the entire 90 ns of classical molecular dynamics (MD) performed; we could observe no significant changes in the most distorted region of the DNA, where the cleavage of phosphodiester bond occurs. In this article, we modeled the reaction mechanism at the interface between Tm1631 and its proposed ligand, the DNA molecule, focusing on cleavage of the phosphodiester bond. After addition of two Mg(2+) ions to the reaction center and extension of classical MD by 50 ns (totaling 140 ns), the DNA ligand stayed bolted to the protein. Results from density functional theory quantum mechanics/molecular mechanics (QM/MM) calculations suggest that the reaction is analogous to known endonuclease mechanisms: an enzyme reaction mechanism with two Mg(2+) ions in the reaction center and a pentacovalent intermediate. The minimum energy pathway profile shows that the phosphodiester bond cleavage step of the reaction is kinetically controlled and not thermodynamically because of a lack of any energy barrier above the accuracy of the energy profile calculation. The role of ions is shown by comparing the results with the reaction mechanisms in the absence of the Mg(2+) ions where there is a significantly higher reaction barrier than in the presence of the Mg(2+) ions.Graphical abstractA protein, Tm1631 from the hyperthermophilic organism Thermotoga maritima belongs to a domain of unknown function protein family. We modeled the reaction mechanism at the interface between Tm1631 and its proposed ligand, the DNA molecule, focusing on cleavage of the phosphodiester bond.
... The presence of a 3'OH…OP β H-bond between the deoxyribose and β-phosphate groups of dNTP has been shown to inversely correlate with k cat in several pol β mutants (i.e., mutants with higher k cat show weaker intramolecular H-bonding) (49). This intramolecular H-bond is present in 99.9% of the configurations sampled in simulations of the GSA complex containing the correct dCTP, but only in 88% of configurations in the PTS model (using a 2.5 Å threshold for the H…O distance as a definition of H-bond, Figure 6). ...
... One indication of such undersolvation is the formation of the ion-pair between the Arg 149 side-chain and the γ-phosphate. This ion-pair was observed in our current and previous (11,49) simulations as well as in MD simulations (52) that employed other force fields, but not in the high-resolution crystal structures (Table 11S). ...
We show how a restricted reaction surface can be used to facilitate the calculation of biologically important contributions of active site geometries and dynamics to DNA polymerase fidelity. Our analysis, using human DNA polymerase beta (pol β), is performed within the framework of an electrostatic linear free energy response (EFER) model. The structure, dynamics, and energetics of pol β-DNA-dNTP interactions are computed between two points on the multidimensional reaction free energy surface. "Point 1" represents a ground state activation intermediate (GSA), which is obtained by deprotonating the terminal 3'OH group of the primer DNA strand. "Point 2" is the transition state (PTS) for the attack of the 3'O(-) (O(nuc)) on the P(α) atom of dNTP substrate, having the electron density of a dianionic phosphorane intermediate. Classical molecular dynamics simulations are used to compute the geometric and dynamic contributions to the formation of right and wrong O(nuc)-P chemical bonds. Matched dCTP·G and mismatched dATP·G base pairs are used to illustrate the analysis. Compared to the dCTP·G base pair, the dATP·G mismatch has fewer GSA configurations with short distances between O(nuc) and P(α) atoms and between the oxygen in the scissile P-O bond (O(lg)) and the nearest structural water. The thumb subdomain conformation of the GSA complex is more open for the mismatch, and the H-bonds in the mispair become more extended during the nucleophilic attack than in the correct pair. The electrostatic contributions of pol β and DNA residues to catalysis of the right and wrong P-O(nuc) bond formation are 5.3 and 3.1 kcal/mol, respectively, resulting in an 80-fold contribution to fidelity. The EFER calculations illustrate the considerable importance of Arg183 and an O(lg)-proximal water molecule to pol β fidelity.
... In the first one, the focus is on the DNA and its behavior to reveal the recognition/binding motifs on the DNA [17] and/or to improve the accuracy in the prediction of binding sites [18]. The second one focuses on the protein structure and how various mutations in proteins or their specific domains affect their ability to bind to the DNA [19][20][21][22]. The third approach focuses on studying the protein-DNA complexes to uncover types of interactions between them and the binding affinity of proteins towards DNA [23][24][25][26]. ...
Our study aims to investigate the impact of the Maturity-onset diabetes of the young 3 disease-linked rSNP rs35126805 located in the HNF-1α gene promotor on the binding of the transcription factor HNF-4α and consequently on the regulation of HNF-1α gene expression. Our focus is to calculate the change in the binding affinity of the transcription factor HNF-4α to the DNA, caused by the regulatory single nucleotide polymorphism (rSNP) through molecular dynamics simulations and thermodynamic analysis of acquired results. Both root-mean-square difference (RMSD) and the relative binding free energy ΔΔGbind reveal that the HNF-4α binds slightly more strongly to the DNA containing the mutation (rSNP) making the complex more stable/rigid, and thereby influencing the expression of the HNF-1α gene. The resulting disruption of the HNF-4α/HNF-1α pathway is also linked to hepatocellular carcinoma metastasis and enhanced apoptosis in pancreatic cancer cells. To the best of our knowledge, this represents the first study where thermodynamic analysis of the results obtained from molecular dynamics simulations is performed to uncover the influence of rSNP on the protein binding to DNA. Therefore, our approach can be generally applied for studying the impact of regulatory single nucleotide polymorphisms on the binding of transcription factors to the DNA.
... Additional support for an allosteric network in pol β comes from MD simulations of cancer-associated mutants with disrupted hydrogen bonding between N279 and a bound dCTP (C = cytosine) that showed strongly attenuated catalytic efficiencies (k cat /K M ) despite the fact that N279 does not make direct contact with the primer or α-phosphate of dCTP (Martinek et al. 2007). Corresponding kinetic studies note the overall change in free energy (ΔG) more closely approximates ΔG of dNTP binding than that of the catalytic reaction (Xiang et al. 2006), supporting a mechanism in which pol β is preorganized for dNTP binding and subsequent allosteric changes propagate to improve or impede pol β catalytic efficiency. ...
Allostery is a ubiquitous biological mechanism in which a distant binding site is coupled to and drastically alters the function of a catalytic site in a protein. Allostery provides a high level of spatial and temporal control of the integrity and activity of biomolecular assembles composed of proteins, nucleic acids, or small molecules. Understanding the physical forces that drive allosteric coupling is critical to harnessing this process for use in bioengineering, de novo protein design, and drug discovery. Current microscopic models of allostery highlight the importance of energetics, structural rearrangements, and conformational fluctuations, and in this review, we discuss the synergistic use of solution NMR spectroscopy and computational methods to probe these phenomena in allosteric systems, particularly protein-nucleic acid complexes. This combination of experimental and theoretical techniques facilitates an unparalleled detection of subtle changes to structural and dynamic equilibria in biomolecules with atomic resolution, and we provide a detailed discussion of specialized NMR experiments as well as the complementary methods that provide valuable insight into allosteric pathways in silico. Lastly, we highlight two case studies to demonstrate the adaptability of this approach to enzymes of varying size and mechanistic complexity.
... The characteristic feature of the active sites of Polβ, tPolλ, and tPolλΔL1 is a network of favorable electrostatic (including hydrogen-bonding) interactions, which interconnects the three apparent binding sites for the nucleobase, dRib, and triphosphate groups of dNTP. 40,63,90,91 These interactions, present already in the RS and preserved during the entire nucleotidyl-transfer reaction, involve the sidechains of Arg149/386/386, Arg183/420/420, and Asn279/513/508, the backbone and sidechain of Tyr271/505/500, and the nucleobase of the templating nucleotide in Polβ/tPolλ/tPolλΔL1. In the solution reaction, the role of the amino acid residues and the templating nucleotide is fulfilled only suboptimally by water molecules, which are incapable of establishing an electrostatically preorganized reaction cage. ...
Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze nucleotidyl-transfer reaction in base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3’OH group of the primer DNA strand, and the subsequent formation and cleavage of P−O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal·mol⁻¹ for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P−O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pKa of the 3’OH group as a predictor of the catalytic rate of X-family DNA polymerases.
... Oelschlaeger et al. developed an efficient method to represent these ions in MD simulations by introducing a dummy atom molecule model rather than the standard ion representation [138]. MD simulations and free energy methods have been employed also to investigate the effects introduced by various mutations of pol [139][140][141]. Pol interacts with APE1 during the BER process. ...
Background:
A major class of chemotherapy drugs targets the genome of cancer cells. These DNA damaging agents induce damage to the DNA helix, resulting in the programmed death of cancer cells. An over-activated DNA repair mechanism in cancer cells can reduce the efficacy of these drugs, thereby eliminating their therapeutic benefit and developing an acquired resistance to these otherwise effective drugs. A promising approach to enhance the therapeutic window of DNA damaging agents is to target the DNA repair pathways causing this type of resistance.
Methods:
Computational approaches have been applied successfully to study many of these DNA repair mechanisms at different scales and focusing on various aspects. The ultimate goal of these studies has been to identify the key players in developing resistance to DNA damaging agents and to design regulators for their activities. This review covers the most important and recent computational efforts toward this goal. This includes modelling the mechanisms involved in DNA repair and identifying novel pharmacological inhibitors for their activities.
Results:
We focus here mainly on the pathways associated with an acquired drug resistance to DNA damaging agents, concentrating on the recent advances in modelling the key mechanisms and foreseeing the future directions in this field.
Conclusions:
We hope that this short, yet comprehensive review can help in discovering novel strategies to overcome the resistance effects inherent in various cancer treatments.
... All complexes obtained from the docking showed between five and nine interprotein ES contacts (ion pairs and hydrogen bonds), but more rigorous treatment of hydrogen bonding across the entire system should verify their stability. 47,78 Figure 3D shows the development of ES contacts between P450 1A2 and cyt b 5 during unconstrained MD simulations of all binding modes evaluated here. Of the interprotein hydrogen bonds of modes A− E, only those in mode A were substantially improved during its ...
Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2–cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2–cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450–cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.
... The wealth of site-directed mutagenesis data (23)(24)(25)(26)(27)(28) on Pol β provides an excellent opportunity to develop robust TCA approaches for prediction of catalytic efficiency of Pol β variants. Recent theoretical studies include the evaluation of contributions of ionized amino acid residues located near Pol β active site on the catalytic efficiency using the semi-microscopic version of Protein Dipoles Langevin dipoles (PDLD/S) method combined with Linear Response Approximation (LRA) method (29), and the identification of the strength of hydrogen bond between N279 and dCTP, as a strong predictor of mutational effects on the catalytic turnover rate for the insertion of dCTP substrate opposite dG (30). ...
We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase β (Pol β). Since the mutated residues in the point-mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol β catalytic site, we assumed that the WT and its point-mutants share the same dianionic phosphorane transition-state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate. Based on this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Ω = (kpol/KD)mut / (kpol/KD)WT using free-energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences. We have performed a pre-steady kinetic analysis for the six mutants for comparison with wild-type Pol β using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Ω values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Ω values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Ω values. These scaled FEP and FEP/LIE methods were also used to predict log Ω for R283A and R283L Pol β mutants.
... which was calculated considering all five DIIIs in the pore, adding up their individual 200 ns, yielding a 1 μs average. If one considers that the configurational activity of this loop is not significantly affected by the neighborhood, the free energy ΔG related to transition from open to narrowed loop, the average rate of occupancy can be estimated by the relation [60,61] The DIII in the five-fold pore configuration, as found on the native DENV surface. In A, the bottom view of the pore, view from inside the virus. ...
We bring to attention a characteristic parasitic pattern present in the dengue virus: it undergoes several intensive thermodynamic variations due to host environmental changes, from a vector's digestive tract, through the human bloodstream and intracellular medium. Comparatively, among the known dengue serotypes, we evaluate the effects that these medium variations may induce to the overall structural characteristics of the Domain III of the envelope (E) protein, checking for stereochemical congruences that could lead to the identification of immunologic relevant regions. We used molecular dynamics and principal component analysis to study the protein in solution, for all four dengue serotypes, under distinct pH and temperature. We stated that, while the core of Domain III is remarkably rigid and effectively unaffected by most of the mentioned intensive variations, the loops account for major and distinguishable flexibilities. Therefore, the rigidity of the Domain III core provides a foothold that projects specifically two of these high flexible loop regions towards the inner face of the envelope pores, which are found at every five-fold symmetry axis of the icosahedron-shaped mature virus. These loops bear a remarkable low identity though with high occurrence of ionizable residues, including histidines. Such stereochemical properties can provide very particular serotype-specific electrostatic surface patterns, suggesting a viral fingerprint region, on which other specific molecules and ions can establish chemical interactions in an induced fit mechanism. We assert that the proposed regions share enough relevant features to qualify for further immunologic and pharmacologic essays, such as target peptide synthesis and phage display using dengue patients' sera.
... Microwave effect of this sort is very similar in nature to the result of immersion of a protein into a less polar and less protic solvent. Consequently, it could also serve to explain why microwaves accelerate S N 2 substitution reactions which favor use of such solvents 57,58 or how microwaves catalyze melting of DNA double-helix 59 through decreased water screening of the repulsive electrostatic interaction 60,61 between the two negatively charged DNA strands and consecutive formation of carcinogenic DNA lesions. 62 However, additional experimental and computational studies are needed in order to confirm or refute this hypothesis. ...
Using molecular dynamics simulations in conjunction with home-developed Split Integration Symplectic Method we effectively decouple individual degrees of freedom of water molecules and connect them to corresponding thermostats. In this way, we facilitate elucidation of structural, dynamical, spectral, and hydration properties of bulk water at any given combination of rotational, translational, and vibrational temperatures. Elevated rotational temperature of the water medium is found to severely hinder hydration of polar molecules, to affect hydration of ionic species in a nonmonotonous way and to somewhat improve hydration of nonpolar species. As proteins consist of charged, polar, and nonpolar amino-acid residues, the developed methodology is also applied to critically evaluate the hypothesis that the overall decrease in protein hydration and the change in the subtle balance between hydration of various types of amino-acid residues provide a plausible physical mechanism through which microwaves enhance aberrant protein folding and aggregation.
... Interestingly, other research has found similar linear relationships in models studying DNA polymerases. Martinek et al. 46 found that the catalytic efficiency of DNA polymerase β correlated linearly with the hydrogen-bond strength between the Asn279 residue and the dCTP substrate, as calculated from molecular dynamics (MD). Zhang et al. 47 compared the efficiency of DNA polymerase with different purine analogs . ...
Human islet amyloid polypeptide (hIAPP) forms cytotoxic fibrils in type-2 diabetes and insulin is known to inhibit formation of these aggregates. In this study, a series of insulin-based inhibitors were synthesized and assessed for their ability to slow aggregation and impact hIAPP-induced membrane damage. Computational studies were employed to examine the underlying mechanism of inhibition. Overall, all compounds were able to slow aggregation at sufficiently high concentrations (10× molar excess); however, only two peptides showed any inhibitory capability at the 1:1 molar ratio (EALYLV and VEALYLV). The results of density functional calculations suggest this is due to the strength of a salt bridge formed with the Arg11 side chain of hIAPP and the inhibitors' ability to span from the Arg11 to past the Phe15 residue of hIAPP, blocking one of the principal amyloidogenic regions of the molecule. Unexpectedly, slowing fibrillogenesis actually increased damage to lipid membranes, suggesting that the aggregation process itself, rather than the fibrilized peptide, may be the cause of cytotoxicity in vivo.
... The Gibbs free energy difference ∆ G BA is then accumulated as a string of increments ∆ G( λ i → λ i + d λ ) obtained from a series of MD simulations performed for the system with parameter λ increasing gradually from 0 to 1 with a small step of d λ (see Figure 3 ). Thus, larger free energy changes must be calculated using N independent MD simulations with different λ values while the free energy difference should be in the order of k B T for each run (73,74). An alternative way to calculate the free energy difference is to employ the thermodynamic integration (TI), which uses the following formula for the Gibbs free energy difference ∆ G BA : ...
The crucial event in the pharmacological action of drugs is the interaction between a drug molecule and its receptor. Molecular recognition and binding affinity of a drug is driven by a flexible steric fit and a molecular field match between two complementary molecular surfaces of the receptor and the approaching ligand. Rational drug design methods, which attempt to predict the free energy change resulting from ligand binding, rely on a detailed knowledge of the physical forces that govern the drug-receptor interactions in several populated configurations of the intermolecular complex. The overall strength of the interaction is determined by the fine balance between the forces contributed by the individual chemical function groups of the two entities and the physiological medium. This paper recounts some basic information about the description of molecular structures and highlights the methods and approximations commonly used to calculate the binding affinity of a drug to its receptor.
... 62 Ranges of reference average backbone torsion angles of canonical A-form RNA structures are listed in Table 1. 63 The conformational free energy difference ∆G freefbound between the TAR-free structure and a bound conformation is determined from a standard thermodynamic relation [64][65][66] according to ...
We report all-atom molecular dynamics and replica exchange molecular dynamics simulations on the unbound human immunodeficiency virus type-1 (HIV-1) transactivation responsive region (TAR) RNA structure and three TAR RNA structures in bound conformations of, in total, approximately 250 ns length. We compare the extent of observed conformational sampling with that of the conceptually simpler and computationally much cheaper constrained geometrical simulation approach framework rigidity optimized dynamic algorithm (FRODA). Atomic fluctuations obtained by replica-exchange molecular dynamics (REMD) simulations agree quantitatively with those obtained by molecular dynamics (MD) and FRODA simulations for the unbound TAR structure. Regarding the stereochemical quality of the generated conformations, backbone torsion angles and puckering modes of the sugar-phosphate backbone were reproduced equally well by MD and REMD simulations, but further improvement is needed in the case of FRODA simulations. Essential dynamics analysis reveals that all three simulation approaches show a tendency to sample bound conformations when starting from the unbound TAR structure, with MD and REMD simulations being superior with respect to FRODA. These results are consistent with the experimental view that bound TAR RNA conformations are transiently sampled in the free ensemble, following a conformation selection model. The simulation-generated TAR RNA conformations have been successfully used as receptor structures for docking. This finding has important implications for RNA-ligand docking in that docking into an ensemble of simulation-generated RNA structures is shown to be a valuable means to cope with large apo-to-holo conformational transitions of the receptor structure.
... Because of its relatively simple structure, it is a popular model enzyme for the larger replicative DNA polymerases. 12 Pol β has previously been used to computationally study the effects of mutations on catalytic efficiencies by calculating the binding free energy of transition state (TS) models (cf. Figure 1) 15,16 , or by finding critical structural parameters that correlate with the observed mutation effects 25 . We have hypothesized that the fit or misfit in the base binding site supports or interferes, respectively, with the chemical reaction in the catalytic site. ...
DNA polymerase beta (pol beta) is a small eukaryotic enzyme with the ability to repair short single-stranded DNA gaps that has found use as a model system for larger replicative DNA polymerases. For all DNA polymerases, the factors determining their catalytic power and fidelity are the interactions between the bases of the base pair, amino acids near the active site, and the two magnesium ions. In this report, we study effects of all three aspects on human pol beta transition state (TS) binding free energies by reproducing a consistent set of experimentally determined data for different structures. Our calculations comprise the combination of four different base pairs (incoming pyrimidine nucleotides incorporated opposite both matched and mismatched purines) with four different pol beta structures (wild type and three mutants). We generate three fragments of the incoming deoxynucleoside 5'-triphosphate-TS and run separate calculations for the neutral base part and the highly charged triphosphate part, using different dielectric constants in order to account for the specific dielectric response. This new approach improves our ability to predict the effect of matched and mismatched base pairing and of mutations in DNA polymerases on fidelity and may be a useful tool in studying the potential of DNA polymerase mutations in the development of cancer. It also supports our point of view with regards to the origin of the structural control of fidelity, allowing for a quantified description of the fidelity of DNA polymerases.
... Additionally, mutation of this residue to valine resulted in a variant with an increased catalytic efficiency due to tighter ground state dNTP binding [67]. The relationship between Asn279 and the incoming nucleotide is important for Pol β activity, as molecular dynamics simulations suggest that the strength of this hydrogen bond correlates with the catalytic efficiency of Pol β [68]. Together, the three residues that contact the incoming dNTP, Phe272, Asp276, and Asn279, are important for both Pol β's activity and fidelity due to their influence on nucleotide binding. ...
The X family of DNA polymerases in eukaryotic cells consists of terminal transferase and DNA polymerases beta, lambda, and mu. These enzymes have similar structural portraits, yet different biochemical properties, especially in their interactions with DNA. None of these enzymes possesses a proofreading subdomain, and their intrinsic fidelity of DNA synthesis is much lower than that of a polymerase that functions in cellular DNA replication. In this review, we discuss the similarities and differences of three members of Family X: polymerases beta, lambda, and mu. We focus on biochemical mechanisms, structural variation, fidelity and lesion bypass mechanisms, and cellular roles. Remarkably, although these enzymes have similar three-dimensional structures, their biochemical properties and cellular functions differ in important ways that impact cellular function.
... The present study focused on the EVB approach and left for a subsequent studies the exploration of the performance of faster but less quantitative approaches, including the LRA, LIE and the PDLD/S-LRA methods [40] that have been found to provide qualitative but physically reasonable trend (e.g [41]), and even ground-state simulations may be useful in the screening process [42]. It is also important to point out that the case of CM is somewhat simpler than cases where there are several transition states with similar energy [43]. ...
The ability of the empirical valence bond (EVB) to be used in screening active site residues in enzyme design is explored in a preliminary way. This validation is done by comparing the ability of this approach to evaluate the catalytic contributions of various residues in chorismate mutase. It is demonstrated that the EVB model can serve as an accurate tool in the final stages of computer-aided enzyme design (CAED). The ability of the model to predict quantitatively the catalytic power of enzymes should augment the capacity of current approaches for enzyme design.
... Additional progress in studies of the fidelity of DNA polymerase has been made by modeling the free energy contributions to the binding of correct and incorrect nucleotides. 12,14,20,21 Further insight has been provided in a recent study, 19 which used computer simulations to model the effect of mutations on the structure of Pol β, and examined the corresponding electrostatic effects by a qualitative analysis. Additionally, theoretical studies of related problems have been reported recently. ...
The relationships between the conformational landscape, nucleotide insertion catalysis and fidelity of DNA polymerase beta are explored by means of computational simulations. The simulations indicate that the transition states for incorporation of right (R) and wrong (W) nucleotides reside in substantially different protein conformations. The protein conformational changes that reproduce the experimentally observed fidelity are significantly larger than the small rearrangements that usually accompany motions from the reactant state to the transition state in common enzymatic reactions. Once substrate binding has occurred, different constraints imposed on the transition states for insertion of R and W nucleotides render it highly unlikely that both transition states can occur in the same closed structure, because the predicted fidelity would then be many orders of magnitude too large. Since the conformational changes reduce the transition state energy of W incorporation drastically they decrease fidelity rather than increase it. Overall, a better agreement with experimental data is attained when the R is incorporated through a transition state in a closed conformation and W is incorporated through a transition state in one or perhaps several partially open conformations. The generation of free energy surfaces for R and W also allow us to analyze proposals about the relationship between induced fit and fidelity.
Exposure to aristolochic acid I and II (AAI and AAII) has been implicated in aristolochic acid nephropathy and urothelial carcinoma. The toxicological effects of AAs are attributed to their ability to form aristolacatam (AL)-purine DNA adducts. Among these lesions, the AL-adenine (ALI-N6-A and ALII-N6-A) adducts cause the 'signature' A→T transversion mutations associated with AA genotoxicity. To provide the currently-missing structural basis for the induction of these signature mutations, the present work uses classical all-atom molecular dynamics (MD) simulations to examine different (i.e., pre-insertion, insertion and post-extension) stages of replication past the most abundant AA adduct (ALI-N6-A) by a representative lesion-bypass DNA polymerase (Dpo4). Our analysis reveals that before dNTP incorporation (i.e., pre-insertion step), ALI-N6-A adopts a nearly-planar conformation at the N6-linkage and the ALI-moiety intercalates within the DNA helix. Since this conformation occupies the dNTP binding site, the same planar lesion conformation results in significant distortion of the polymerase active site at the insertion step and therefore replication will likely not be successful. However, if ALI-N6-A undergoes a small conformational change to introduce nonplanarity at the N6-linkage during the insertion step, minimal distortion occurs in the Dpo4 active site upon incorporation of dATP. This insertion and subsequent extension would initially lead to A:A mismatches, and then result in A→T transversion mutations during the second round of replication. In contrast, if a large conformation flip of the ALI-moiety occurs at the insertion step to reorient the bulky moiety from an intercalated position into the major groove, (non-mutagenic) dTTP incorporation will be favored. MD simulations on post-extension complexes reveal that damaged DNA will likely further rearrange during later replication steps to acquire a base-displaced intercalated conformation that is similar to that previously reported for (unbound) ALI-N6-A adducted DNA, with the exception of slight nonplanarity at the lesion site. Overall, our results provide a structural explanation for both the successful non-mutagenic lesion bypass and the preferential misincorporation of dATP opposite ALI-N6-A, and thereby rationalize the previously-reported induction of A→T signature transversion mutations associated with AAs. This work should thereby inspire future biochemical experiments and modeling studies on the replication of this important class of DNA lesions by related human translesion synthesis polymerases.
Acrylonitrile (AN) is widely used in the manufacture of resins, plastics and polymers, where workers are exposed to it during its production, transportation and application. After intake a portion of AN is converted to cyanoethylene oxide (CEO) by cytochrome P450 2E1. Both AN and CEO represent possible chemical carcinogens leading to DNA damage mainly in the form of the major 7-(2-oxoethyl)deoxyguanosine adduct. A kinetic model for its formation was devised and a corresponding second-order rate constant obtained from the experimental data on the reaction with CEO. Then a series of \textit{ab initio}, density functional theory and semiempirical calculations of activation free energies was performed on the alkylation of nucleic bases with both CEO and AN. The combination of Hartree-Fock level of theory with the flexible 6-311++G(d,p) basis set and Langevin dipoles implicit solvation model gave the best agreement with the experimental activation barrier. It also predicted relative reactivities of all four nucleobases that are in agreement with the experimentally reported adduct yields. Moreover, this combination predicted higher reactivity of CEO than AN with all four nucleobases corroborating the experimental hypothesis that SN2 substitution of CEO rather than direct Michael addition of AN is responsible for the genotoxic properties of AN. Last but not least, in a broader context this paper points to the applicability of quantum-chemical methods to the studies of carcinogenesis.
DNA Polymerase β (Pol β) repairs single-nucleotide gapped DNA (sngDNA) by enzymatic incorporation of the Watson-Crick partner nucleotide at the gapped position opposite the templating nucleotide. The process by which matching nucleotide is incorporated into a sngDNA sequence has been relatively well-characterized, but the process of discrimination from nucleotide misincorporation remains unclear. We report here NMR spectroscopic characterization of full-length, uniformly labeled Pol β in apo, sngDNA-bound binary, and ternary complexes containing matching and mismatching nucleotide. Our data indicate that, while binding of the correct nucleotide to the binary complex induces chemical shift changes consistent with the process of enzyme closure, the ternary Pol β complex containing a mismatching nucleotide exhibits no such changes and appears to remain in an open, unstable, binary-like conformation. Our findings support an induced-fit mechanism for polymerases in which a closed ternary complex can only be achieved in the presence of matching nucleotide.
Given the increasing complexity of simulated molecular systems, and the fact that simulation times have now reached milliseconds to seconds, immense amounts of data (in the gigabyte to terabyte range) are produced in current molecular dynamics simulations. Manual analysis of these data is a very time-consuming task, and important events that lead from one intermediate structure to another can become occluded in the noise resulting from random thermal fluctuations. To overcome these problems and facilitate a semi-automated data analysis, we introduce in this work a measure based on C(α) torsion angles: torsion angles formed by four consecutive C(α) atoms. This measure describes changes in the backbones of large systems on a residual length scale (i.e., a small number of residues at a time). Cluster analysis of individual C(α) torsion angles and its fuzzification led to continuous time patches representing (meta)stable conformations and to the identification of events acting as transitions between these conformations. The importance of a change in torsion angle to structural integrity is assessed by comparing this change to the average fluctuations in the same torsion angle over the complete simulation. Using this novel measure in combination with other measures such as the root mean square deviation (RMSD) and time series of distance measures, we performed an in-depth analysis of a simulation of the open form of DNA polymerase I. The times at which major conformational changes occur and the most important parts of the molecule and their interrelations were pinpointed in this analysis. The simultaneous determination of the time points and localizations of major events is a significant advantage of the new bottom-up approach presented here, as compared to many other (top-down) approaches in which only the similarity of the complete structure is analyzed.
The outbreak of avian influenza A (H5N1) virus has raised a global concern for both the animal as well as human health. Besides vaccination, that may not achieve full protection in certain groups of patients, inhibiting neuraminidase or the transmembrane protein M2 represents the main measure of controlling the disease. Due to alarming emergence of influenza virus strains resistant to the currently available drugs, development of new neuraminidase N1 inhibitors is of utmost importance. The present paper provides an overview of the recent advances in the design of new antiviral drugs against avian influenza. It also reports findings in binding free energy calculations for nine neuraminidase N1 inhibitors (oseltamivir, zanamivir, and peramivir -carboxylate, -phosphonate, and -sulfonate) using the Linear Interaction Energy method. Molecular dynamics simulations of these inhibitors were performed in a free and two bound states - the so called ˝open˝ and ˝closed˝ conformations of neuraminidase N1. Obtained results successfully reproduce the experimental binding affinities of the already known neuraminidase N1 inhibitors, i.e. peramivir being a stronger binder than zanamivir that is in turn stronger binder than oseltamivir, or phosphonate inhibitors being stronger binders than their carboxylate analogues. In addition, the newly proposed sulfonate inhibitors are predicted to be the strongest binders - a fact to be confirmed by their chemical synthesis and a subsequent test of their biological activity. Finally, contributions of individual inhibitor moieties to the overall binding affinity are explicitly evaluated to assist further drug development towards inhibition of the H5N1 avian influenza A virus.
Objectives:
The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Human cytochrome P450 (CYP) 1A1 and 1A2 enzymes were found to be responsible for the AAI reductive activation to form AAI-DNA adducts, while its structurally related analogue, CYP1B1 is almost without such activity. However, knowledge of the differences in mechanistic details of CYP1A1-, 1A2-, and 1B1- mediated reduction is still lacking. Therefore, this feature is the aim of the present study.
Methods:
Molecular modeling capable of evaluating interactions of AAI with the active site of human CYP1A1, 1A2 and 1B1 under the reductive conditions was used. In silico docking, employing soft-soft (flexible) docking procedure was used to study the interactions of AAI with the active sites of these human enzymes.
Results:
The predicted binding free energies and distances between an AAI ligand and a heme cofactor are similar for all CYPs evaluated. AAI also binds to the active sites of CYP1A1, 1A2 and 1B1 in similar orientations. The carboxylic group of AAI is in the binding position situated directly above heme iron. This ligand orientation is in CYP1A1/1A2 further stabilized by two hydrogen bonds; one between an oxygen atom of the AAI nitro-group and the hydroxyl group of Ser122/Thr124; and the second bond between an oxygen atom of dioxolane ring of AAI and the hydroxyl group of Thr497/Thr498. For the CYP1B1:AAI complex, however, any hydrogen bonding of the nitro-group of AAI is prevented as Ser122/Thr124 residues are in CYP1B1 protein replaced by hydrophobic residue Ala133.
Conclusion:
The experimental observations indicate that CYP1B1 is more than 10× less efficient in reductive activation of AAI than CYP1A2. The docking simulation however predicts the binding pose and binding energy of AAI in the CYP1B1 pocket to be analogous to that found in CYP1A1/2. We believe that the hydroxyl group of S122/T124 residue, with its polar hydrogen placed close to the nitro group of the substrate (AAI), is mechanistically important, for example it could provide a proton required for the stepwise reduction process. The absence of a suitable proton donor in the AAI-CYP1B1 binary complex could be the key difference, as the nitro group is in this complex surrounded only by the hydrophobic residues with potential hydrogen donors not closer than 5 Å.
Change in pH plays crucial role in stability and function of dengue envelope (DENV) protein during conformational transition from dimeric (pre-fusion state) to trimeric form (post-fusion state). In the present study we have performed various molecular dynamics (MD) simulations of trimeric DENV protein at different pH and ionic concentrations. We have used total binding energy to justify the stability of complex using MMPBSA method. We found a remarkable increase in stability of complex at neutral pH (pH~7) due to the increment of sodium ions. However, at very low pH (pH~4), total energy of the complex becomes high enough to destabilize the complex. At a specific pH, almost at range of 6, the stability of the complex is significantly better than stability of trimer at neutral pH, which connotes that trimer is most stable at this pH (pH ~6).
To investigate whether an open-to-closed transition before the chemical step and induced-fit mechanism exist in DNA polymerase μ (pol μ), we analyze a series of molecular-dynamics simulations with and without the incoming nucleotide in various forms, including mutant systems, based on pol μ's crystal ternary structure. Our simulations capture no significant large-scale motion in either the DNA or the protein domains of pol μ. However, subtle residue motions can be distinguished, specifically of His(329) and Asp(330) to assemble in pol μ's active site, and of Gln(440) and Glu(443) to help accommodate the incoming nucleotide. Mutant simulations capture a DNA frameshift pairing and indicate the importance of Arg(444) and Arg(447) in stacking with the DNA template, and of Arg(448) and Gln(440) in helping to stabilize the position of both the DNA template and the incoming nucleotide. Although limited sampling in the molecular-dynamics simulations cannot be ruled out, our studies suggest an absence of a large-scale motion in pol μ. Together with the known crystallization difficulties of capturing the open form of pol μ, our studies also raise the possibility that a well-defined open form may not exist. Moreover, we suggest that residues Arg(448) and Gln(440) may be crucial for preventing insertion frameshift errors in pol μ.
The insertion of a DNA base moiety at the end of a DNA duplex to form a Watson-Crick or wobble pair during DNA annealing or replication is a step of fundamental biological importance. Therefore, we investigated the energetics of a formation of the terminal G x C, G x T, and G x A base pairs in DNA containing a 5'-dangling G adjacent to the base insertion point using differential scanning calorimetry and computer simulations. The energies calculated along classical molecular dynamics trajectories in aqueous solution were analyzed in the framework of linear-response approximation (LRA) to obtain relative free energies for the base insertion and their electrostatic, van der Waals, and preorganization components. Using the generic set of LRA parameters, the calculated free energies disfavored the mispair formation by 2.5 (G x C --> G x T) and 1.7 (G x C --> G x A) kcal/mol, in reasonable agreement with the experimental free energy differences of 1.8 and 1.4 kcal/mol, respectively. The calculated preorganization components of these free energies of 0.6 (G x C --> G x T) and -0.1 (G x C --> G x A) kcal/mol show that electrostatic preorganization, which is an important source of DNA replication fidelity, plays a lesser role in the mispair destabilization in the absence of DNA polymerase.
The outbreak of avian influenza A subtype H5N1 virus has raised a global concern for both animal as well as human health. Recently, drug resistance in H5N1 infections has been widely reported due to neuraminidase mutations. Consequently, the understanding of inhibitor-neuraminidase interactions at the molecular level represents the main goal of our study. Molecular dynamics simulations were carried out for the neuraminidase N1 in complex with six inhibitors--oseltamivir, zanamivir, peramivir, and their phosphonate analogues. Molecular dynamics trajectories were extensively analyzed in terms of important interactions between inhibitors and the enzyme target. Results show that open and closed forms (defined by the relative position of the flexible 150-loop) of neuraminidase N1 interchange during the course of 20 ns molecular dynamics simulation of the protein-inhibitor complexes. Reported free energies of closing indicate that the carboxylate inhibitors prefer the closed form more than their phosphonate analogues. This can be understood in view of the negative total charge (-1 e0) of the phosphonate inhibitors, which repels the Asp151 residue of the loop away from the inhibitor and drives the complex into the open form. Obtained results constitute new valuable information to assist further drug development of inhibitors against the H5N1 avian influenza A virus and could also inspire similar studies for other systems of the influenza family such as the 2009 influenza A (H1N1) virus.
The mechanism of DNA polymerase beta-catalyzed nucleotidyl transfer consists of chemical steps involving primer 3' OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. Ambiguity in the nature of the rate-limiting step and active-site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Analogues of dGTP where the beta-gamma bridging oxygen is replaced with fluorine-substituted methylene groups have been shown to probe the contribution of leaving-group elimination to the overall catalytic rate (Biochemistry 46, 461-471). Here, the analysis is expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogues opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogues, with magnitude dependent on the identity of the nascent base pair. This observation, and the absence of an analogous effect on ground state analogue binding (Kd values), points to active-site structural differences at the chemical transition state. Reduced catalysis with bulky halo-containing substrates is manifested in the fidelity of T-G incorporation, where the CCl2-bridging analogue shows a 27-fold increase in fidelity over the natural dGTP. Solvent pH and deuterium isotope-effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation.
The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase β (β-pol) were
labeled using a photoreactive analog of dCTP, exo-N-[β-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine-5′-triphosphate (FABdCTP). Two approaches of photolabeling were utilized.
In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme
and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently,
radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3′-end of the radiolabeled
primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the
intensity of cross-linking was observed with either approach. FABdCTP cross-linking was increased in the presence of a dideoxyterminated
primer-template with RT, but not with β-pol, suggesting a significant influence of prior primer-template binding on dNTP binding
for RT. Mutagenesis of β-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary
complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of
the dNTP binding pocket.
In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and
Arg283 of DNA polymerase β are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5′-triphosphate (dNTP),
the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson,
S. H., and Kraut, J.(1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was
expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and
catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess
the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or
histidine did not influence catalytic efficiency
(kcat/Km) or fidelity. The hydrogen bonding potential between the side chain of Asn279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency
was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased
dramatically for all mutants of Arg283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 5000-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from
short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant
were similar and indicated misincorporations resulting in frequent T•dGTP and A•dGTP mispairing. With R283A, a dGMP was incorporated
opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant
of Arg283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase β and
the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and
nucleotide discrimination.
The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (A.T) or incorrect (G.T, C.T, T.T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon dissociation (delta delta Go) are only 0.2, 0.3, and 0.4 kcal.mol-1 (1 cal = 4.18 J) for terminal A.T compared to G.T, C.T, and T.T mispairs, respectively, at 37 degrees C. We show that enthalpy changes are directly correlated with entropy changes for normal and abnormal base pairs in DNA in aqueous solution and that delta delta Go values are small because of near cancellation of corresponding enthalpy and entropy components. The kinetics of elongating primer termini are measured with purified Drosophila DNA polymerase alpha. The matched A.T terminus is found to be extended approximately 200 times faster than a G.T mismatch and 1400 and 2500 times faster than C.T and T.T mismatches, respectively. Enzymatic discrimination against elongating mismatched termini is based mainly on Km rather than Vmax differences. From Km at 37 degrees C, we find delta delta Go values of 2.6-3.7 kcal.mol-1, about an order of magnitude greater than indicated by melting data. A similar measurement of nucleotide insertion kinetics has previously found rates of forming A.T base pairs to be 500 times greater than G.T mispairs and 20,000 times greater than C.T and T.T mispairs. Here also, Km differences are mainly responsible for discrimination and indicate even larger delta delta Go values (4.3-4.9 kcal.mol-1). Thus, free energy differences between correct and incorrect base pairs in the active site cleft of polymerase appear to be greater than 10 times as large as in aqueous medium. We explore the idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalpy differences sufficiently to account for nucleotide insertion and extension fidelity.
The origin of the catalytic power of enzymes is discussed, paying attention to evolutionary constraints. It is pointed out that enzyme catalysis reflects energy contributions that cannot be determined uniquely by current experimental approaches without augmenting the analysis by computer simulation studies. The use of energy considerations and computer simulations allows one to exclude many of the popular proposals for the way enzymes work. It appears that the standard approaches used by organic chemists to catalyze reactions in solutions are not used by enzymes. This point is illustrated by considering the desolvation hypothesis and showing that it cannot account for a large increase in kcat relative to the corresponding kcage for the reference reaction in a solvent cage. The problems associated with other frequently invoked mechanisms also are outlined. Furthermore, it is pointed out that mutation studies are inconsistent with ground state destabilization mechanisms. After considering factors that were not optimized by evolution, we review computer simulation studies that reproduced the overall catalytic effect of different enzymes. These studies pointed toward electrostatic effects as the most important catalytic contributions. The nature of this electrostatic stabilization mechanism is far from being obvious because the electrostatic interaction between the reacting system and the surrounding area is similar in enzymes and in solution. However, the difference is that enzymes have a preorganized dipolar environment that does not have to pay the reorganization energy for stabilizing the relevant transition states. Apparently, the catalytic power of enzymes is stored in their folding energy in the form of the preorganized polar environment.
Structures of DNA polymerase (pol) beta bound to single-nucleotide gapped DNA had revealed that the lyase and pol domains form a "doughnut-shaped" structure altering the dNTP binding pocket in a fashion that is not observed when bound to non-gapped DNA. We have investigated dNTP binding to pol beta-DNA complexes employing steady-state and pre-steady-state kinetics. Although pol beta has a kinetic scheme similar to other DNA polymerases, polymerization by pol beta is limited by at least two partially rate-limiting steps: a conformational change after dNTP ground-state binding and product release. The equilibrium binding constant, K(d)((dNTP)), decreased and the insertion efficiency increased with a one-nucleotide gapped DNA substrate, as compared with non-gapped DNA. Valine substitution for Asp(276), which interacts with the base of the incoming nucleotide, increased the binding affinity for the incoming nucleotide indicating that the negative charge contributed by Asp(276) weakens binding and that an interaction between residue 276 with the incoming nucleotide occurs during ground-state binding. Since the interaction between Asp(276) and the nascent base pair is observed only in the "closed" conformation of pol beta, the increased free energy in ground-state binding for the mutant suggests that the subsequent rate-limiting conformational change is not the "open" to "closed" structural transition, but instead is triggered in the closed pol conformation.
Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, alpha-helix N of DNA polymerase beta is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.
Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a "simplified" model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.
The initial electron-transfer reaction in photosynthetic bacterial reaction centers is the transfer of an electron from the excited state (P{sup *}) of a reactive bacteriochlorophyll dimer (P) to a bacteriopheophytin (H), generating a P{sup +}H{sup -} radical pair. In this investigation we have shown that models with proper dielectric boundaries lead to calculated electrostatic energies that are relatively insensitive to the assumptions made concerning the solvent and the charges assigned to ionized groups of the protein. Using such models leads to good agreement with the experimentally measured energy of the relaxed P{sup +}H{sup -} radical pair and places P{sup +}B{sup -}H close to P{sup *} in energy. By contrast, treatments that neglect the self-energies of the electron carriers and do not account for the screening of the field of the ionized residues place P{sup +}B{sup -}H substantially above P{sup *}. 35 refs., 5 figs., 9 tabs.
Consistent approaches for calculations of solvation free energies should provide results which are independent of the size of the simulation region. Simulations that use periodic boundary conditions and a standard Ewald treatment yield size dependent results. Corrections that can overcome this problem have been formulated, but have not yet been fully validated for solutes with general charge distributions. Furthermore, Ewald treatments of proteins may involve size dependent problems whose nature has not been explored by systematic studies. Here we demonstrate that our surface constraint all-atom solvent (SCAAS) model with its spherical boundary conditions (that include special polarization constraints) provides proper size independent results. It is also pointed out that this approach lends itself to an effective treatment of long-range interactions and offers a useful way of obtaining size independent free energies in studies of electrostatic effects in proteins. © 1998 American Institute of Physics.
A consistent simulation of ionic or strongly polar solutes in polar solvents presents a major challenge from both fundamental and practical aspects. The frequently used method of periodic boundary conditions (PBC) does not correctly take into account the symmetry of the solute field. Instead of using PBC, it is natural to model this type of system as a sphere (with the solute at the origin), but the boundary conditions to be used in such a model are not obvious. Early calculations performed with our surface constrained soft sphere dipoles (SCSSD) model indicated that the dipoles near the surface of the sphere will show unusual orientational preferences (they will overpolarize) unless a corrective force is included in the model, and thus we implemented polarization constraints in this spherical model of polar solutions. More recent approaches that treated the surface with stochastic dynamics, but did not take into account the surface polarization effects, were also found to exhibit these nonphysical orientational preferences. The present work develops a surface constrained all-atom solvent (SCAAS) model in order to consistently treat the surface polarization effects in all-atom molecular dynamics simulations. The SCAAS model, which was presented in a preliminary way in previous works, introduces surface constraints as boundary conditions in order to make the necessarily finite system behave as if it was part of an infinite system. The performance of the model with regard to various properties of bulk water is examined by comparing its results to those obtained by PBC simulations. The results obtained from SCAAS models of different sizes are found to be similar to each other and to the corresponding PBC results. The performance of the model in simulations of solvated ions is emphasized and a comparison of the results obtained with spheres of different sizes demonstrates that the model does not possess significant size dependence. This indicates that the model can be used with a relatively small number of solvent molecules for convergent simulation of structure, energetics, and dynamics of polar solutions. The much simpler fixed
center Langevin dipoles (FCLD) model is also examined and found to provide a
powerful tool for estimating solvation free energies. Finally, a preliminary
study of the dielectric properties of the SCAAS model is reported and the potential of this model for exploring the correct implementation of the solvent reaction field is discussed.
We present a theoretical study of the selection of right/wrong dNTP substrates by DNA polymerases at the initial binding step, a major component of the DNA replication fidelity. Linear-response analysis (LRA) and molecular dynamics simulations are performed starting from the X-ray crystal structure of a ternary DNA polymerase β ·DNA · ddCTP complex. These simulations provide converged structures of ternary complexes containing all four Watson−Crick (W−C) pairs as well as 11 neutral mismatched dNTP-template base pairs in the anti−anti conformations. The signs and overall magnitudes of the calculated relative free energies for binding of each dNTP to pol β ·DNA complexes, which contain either correct or incorrect templating bases, agree with the observed universal preference of DNA polymerases for W−C base pairs. Overall, the binding free-energy differences of each dNTP to right versus wrong templates are found to be dominated by electrostatic interactions between templating and dNTP bases. However, about half of the electrostatic contribution can be attributed to the steric preorganization of the polymerase active site that was generated by the protein folding process. The preorganized site maintains optimal W−C pairing for matched bases while forcing mismatched pairs into configurations far from their ideal gas-phase geometries. Consequently, the preorganized site is responsible for large template contributions to fidelity. Individual additive contributions to fidelity are determined for active site residues. Interactions between incoming dNTPs and Asn279 and Tyr262 protein residues contribute significantly to the binding component of fidelity, with the Asn279 residue most effective in destabilizing each of the 15 nucleotide mispairs in neutral anti−anti conformations. Active site amino acids can also exert deleterious effects on fidelity. Tyr262 enhances base substitution fidelity via mispair destabilization, but it also stabilizes slipped mispaired primer·template structures that are potential precursors for +1 frameshift mutations. The inclusion of an extra water molecule in the active cleft was found to stabilize several wobble base pairs. Calculations performed at this level of “fine” detail are used to predict the effects of amino acid substitutions on the fidelity for mutant forms of pol β, thus providing a deeper understanding of the role of the polymerase active site in ensuring replication accuracy.
The initial electron-transfer reaction in photosynthetic bacterial reaction centers is the transfer of an electron from the excited state (P{sup *}) of a reactive bacteriochlorophyll dimer (P) to a bacteriopheophytin (H), generating a P{sup +}Hâ» radical pair. In this investigation we have shown that models with proper dielectric boundaries lead to calculated electrostatic energies that are relatively insensitive to the assumptions made concerning the solvent and the charges assigned to ionized groups of the protein. Using such models leads to good agreement with the experimentally measured energy of the relaxed P{sup +}Hâ» radical pair and places P{sup +}Bâ»H close to P{sup *} in energy. By contrast, treatments that neglect the self-energies of the electron carriers and do not account for the screening of the field of the ionized residues place P{sup +}Bâ»H substantially above P{sup *}. 35 refs., 5 figs., 9 tabs.
Recently, evidence has accumulated that mutations in DNA repair genes might be associated with certain steps in carcinogenesis. The DNA polymerase gene is one of the DNA repair genes, and mutations in it have been detected in 83% of human colorectal cancers. To assess the involvement of polymerase gene mutations in the development of human prostate cancers, we performed sequence analyses of human DNA samples. Unexpectedly, we found six regions that were polymorphic. This information should be taken into consideration at the time of sequence analysis of the DNA polymerase gene.s
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
One of the major problems in molecular-dynamics simulations of polar fluids -or macromolecular systems is the evaluation of electrostatic interactions. A system of N atoms demands an amount of work proportional to N2 for such calculations. Truncation procedures that neglect a significant part of the long-range effects are often necessary for computational feasibility, though such procedures may introduce serious errors in the simulations. This work introduces a simple and very effective approach for treating the long-range electrostatic forces. The present method, which is referred to as the local reaction field method, follows some of the ideas of the previously developed generalized Ewald method but then develops into a much simpler method. This is done by dividing the system into M groups of atoms and evaluating separately the short- and long-range contributions to the potential of each group. The short-range potential is evaluated explicitly as in any standard truncation method, while the long-range potential is approximated by the first four terms in a multipole expansion. In addition, the long-range potential is updated only once in every L time steps leading to a method of the order of N X M X q/L + N X P where q is related to the number of expansion terms and P is the average number of atoms inside the cutoff range. The speed, accuracy, and precision of the present method is assessed by evaluating the self-energy of a sodium ion in water and the self-energies of the acidic residues of bovine pancreatic trypsin inhibitor using an adiabatic charging free-energy perturbation approach. It is found that the present method reproduces accurately the corresponding results obtained without any cutoff but an order of magnitude faster. Furthermore, at the limit of very large systems, the speed of this method can be 2 orders of magnitude faster than that of the no-cutoff method even when each group contains only a small number of atoms. It is also found that the method gives much better results for electrostatic energies in proteins than those obtained by truncation methods. The stability and speed of the local reaction field method provides a powerful tool for the microscopic evaluation of electrostatic energies in macromolecules.
We present the derivation of a new molecular mechanical force field for simulating the structures, conformational energies, and interaction energies of proteins, nucleic acids, and many related organic molecules in condensed phases. This effective two-body force field is the successor to the Weiner et al, force field and was developed with some of the same philosophies, such as the use of a simple diagonal potential function and electrostatic potential fit atom centered charges. The need for a 10-12 function for representing hydrogen bonds is no longer necessary due to the improved performance of the new charge model and new van der Waals parameters. These new charges are determined using a 6-31G basis set and restrained electrostatic potential (RESP) fitting and have been shown to reproduce interaction energies, free energies of solvation, and conformational energies of simple small molecules to a good degree of accuracy. Furthermore, the new RESP charges exhibit less variability as a function of the molecular conformation used in the charge determination. The new van der Waals parameters have been derived from liquid simulations and include hydrogen parameters which take into account the effects of any geminal electronegative atoms. The bonded parameters developed by Weiner et al. were modified as necessary to reproduce experimental vibrational frequencies and structures. Most of the simple dihedral parameters have been retained from Weiner et. al., but a complex set of phi and psi parameters which do a good job of reproducing the energies of the low-energy conformations of glycyl and alanyl dipeptides has been developed for the peptide backbone.
Quantitative studies of the energetics of enzymatic reactions and the corresponding reactions in aqueous solutions indicate that charge stabilization is the most important energy contribution in enzyme catalysis. Low electrostatic stabilization in aqueous solutions is shown to be consistent with surprisingly large electrostatic stabilization effects in active sites of enzymes. This is established quantitatively by comparing the relative stabilization of the transition states of the reaction of lysozyme and the corresponding reaction is aqueous solution.
Recently, evidence has accumulated that mutations in DNA repair genes might be associated with certain steps in carcinogenesis. The DNA polymerase beta gene is one of the DNA repair genes, and mutations in it have been detected in 83% of human colorectal cancers. To assess the involvement of polymerase beta gene mutations in the development of human prostate cancers, we performed sequence analyses of human DNA samples. Unexpectedly, we found six regions that were polymorphic. This information should be taken into consideration at the time of sequence analysis of the DNA polymerase beta gene.
We have analysed the frequency with which potential hydrogen bond donors and acceptors are satisfied in protein molecules. There are a small percentage of nitrogen or oxygen atoms that do not form hydrogen bonds with either solvent or protein atoms, when standard criteria are used. For high resolution structures 9.5% and 5.1% of buried main-chain nitrogen and oxygen atoms, respectively, fail to hydrogen bond under our standard criteria, representing 5.8% and 2.1% of all main-chain nitrogen and oxygen atoms. We find that as the resolution of the data improves, the percentages fall. If the hydrogen bond criteria are relaxed many of these unsatisfied atoms form weak hydrogen bonds. However, there remain some buried atoms (1.3% NH and 1.8% CO) that fail to hydrogen bond without any immediately obvious compensating interactions.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
The kinetic parameters (kpol, Kd app) for all possible correct and incorrect pairing between the A, T, G, and C bases were determined for wild-type (WT) rat DNA polymerase beta (pol beta) and the R283A mutant under pre-steady-state kinetic assay conditions. The base substitution fidelities of these two proteins were then determined for all 12 possible mispairs representing the first complete fidelity analysis of polymerases using pre-steady-state kinetics. The results led to several significant findings: (i) For both WT and R283A, the fidelity is determined primarily by kpol (decreases for the incorporation of incorrect nucleotides) and to a small extent by Kd app (increases for the incorporation of incorrect nucleotides). (ii) In general, the fidelity for the Y.X (incorporation of dXTP opposite template dYMP) mismatch is different from that for the X.Y mismatch, reflecting the asymmetry of the active site. (iii) The fidelity of R283A is reduced in all 12 mispairs compared to that of WT. The extent of decrease varies from 200-fold for the A.G mispair to 2.5-fold for the T.C mispair. In general, the differences in fidelity between the mutant and WT are greater for purine.purine mismatches (up to 200-fold) than purine.pyrimidine, pyrimidine. purine, or pyrimidine.pyrimidine mismatches (up to 19-fold). (iv) Overall, the decreases in the fidelity of the R283A mutant are caused mainly by changes in the values of kpol; the kpol values of correct incorporations decrease to a greater extent for the R283A mutant with respect to WT than those of incorrect incorporations. With the exception of G.C, the values of Kd app for the WT and R283A mutant remain constant for correct pairings and vary by less than a factor of 4 for incorrect pairings. (v) For WT pol beta, the Kd app of G.C (8.6 microM) is distinctly smaller than that of other correct base pairs (41-108 microM). For the R283A mutant, the kpol of G.C is higher by a factor of 15-17.
DNA polymerase beta (pol beta) from rat brain, overexpressed in Escherichia coli, was used as a model to study the factors responsible for substrate specificity [kpol, Kd(app) and kpol/Kd(app)] and fidelity during DNA synthesis. The roles of two active-site residues, Asn-279 and Tyr-271, were examined by construction of N279A, N279Q, Y271A, Y271F and Y271S mutants followed by structural analyses by NMR and CD and functional analyses by pre-steady-state kinetics. The results are summarized as follows. (i) None of the two-dimensional NMR spectra of the mutants was significantly perturbed relative to that for wild-type pol beta, suggesting that Tyr-271 and Asn-279 are not important for the global structure of the protein. (ii) CD analyses of guanidinium hydrochloride-induced denaturation showed that all mutants behaved similarly to the wild type in the free energy of denaturation, suggesting that Tyr-271 and Asn-279 are not critical for the conformational stability of pol beta. (iii) The Kd(app) for the correct dNTP was lower than that for the incorrect dNTP by a factor of 10-30 in the case of wild-type pol beta. Upon mutation to give N279A and N279Q, the Kd(app) for the correct dNTP increased by a factor of 15-25. As a consequence, the Kd(app) values for the correct and incorrect nucleotides were similar for N279A and N279Q, suggesting that the main function of the side chain of Asn-279 is in discrimination between the binding of correct and incorrect dNTPs. (iv) In the case of the Y271A mutant, the fidelity and the catalytic efficiency kpol/Kd(app) were little perturbed relative to the wild type. However, both the kpol and Kd(app) values for dNTP were 4-8 times lower in the case of the Y271A mutant than the corresponding values for wild-type pol beta. Since the chemical step may not be rate-limiting for wild-type pol beta, the effect on kpol could be quite significant if it is caused by a perturbation in the chemical step. (v) Pol beta displayed the greatest specificity towards the G:C base pair, which is incorporated during base excision repair of G:U and G:T mispairs. This specificity was slightly enhanced for the Y271F mutant.
A long-standing question in cancer biology has been the extent to which DNA repair may be altered during the process of carcinogenesis. We have shown recently that DNA polymerase beta (beta-pol) provides a rate-determining function during in vitro repair of abasic sites by one of the mammalian DNA base excision repair pathways. Therefore, altered expression of beta-pol during carcinogenesis could alter base excision repair and, consequently, be critical to the integrity of the mammalian genome. We examined the expression of beta-pol in several cell lines and human adenocarcinomas using a quantitative immunoblotting method. In cell lines from normal breast or colon, the level of beta-pol was approximately 1 ng/mg cell extract, whereas in all of the breast and colon adenocarcinoma cell lines tested, a higher level of beta-pol was observed. In tissue samples, colon adenocarcinomas had a higher level of beta-pol than adjacent normal mucosa. Breast adenocarcinomas exhibited a wide range of beta-pol expression: one tumor had a much higher level of beta-pol (286 ng/mg cell extract) than adjacent normal breast tissue, whereas another tumor had the same level of beta-pol as adjacent normal tissue. Differences in beta-pol expression level, from normal to elevated, were also observed with prostate adenocarcinomas. All kidney adenocarcinomas tested had a slightly lower beta-pol level than adjacent normal tissue. This study reveals that the base excision repair enzyme DNA polymerase beta is up-regulated in some types of adenocarcinomas and cell lines, but not in others.
A new molecular dynamics program for free energy calculations in biomolecular systems is presented. It is principally designed for free energy perturbation simulations, empirical valence bond calculations, and binding affinity estimation by linear interaction energy methods. Evaluation of ligand-binding selectivity and free energy profiles for nucleophile activation in two protein tyrosine phosphatases as well as absolute binding affinity estimation for a lysine-binding protein are given as examples.
The specific catalytic roles of two groups of DNA polymerase beta active site residues identified from crystal structures were investigated: residues possibly involved in DNA template positioning (Lys280, Asn294, and Glu295) and residues possibly involved in binding the triphosphate moiety of the incoming dNTP (Arg149, Ser180, Arg183, and Ser188). Eight site-specific mutants were constructed: K280A, N294A, N294Q, E295A, R149A, S180A, R183A, and S188A. Two-dimensional NMR analysis was employed to show that the global conformation of the mutants has not been perturbed significantly. Pre-steady-state kinetic analyses with single-nucleotide gapped DNA substrates were then performed to obtain the rate of catalysis at saturating dNTP (k(pol)), the apparent dissociation constant for dNTP (K(d)), catalytic efficiency k(pol)/K(d), and fidelity. Of the three template-positioning residues, Asn294 and Glu295 (but not Lys280) contribute significantly to k(pol). Taken together with other data, the results suggest that these two residues help to stabilize the transition state during catalysis even though they interact with the DNA template backbone rather than directly with the incoming dNTP or the opposite base on the template. Furthermore, the fidelity increases by up to 19-fold for N294Q due to differential k(pol) effects between correct and incorrect nucleotides. Of the four potential triphosphate-binding residues, Ser180 and Arg183 contribute significantly to k(pol) while the effects of R149A are relatively small and are primarily on K(d), and Ser188 appears to play a minimal role in the catalysis by Pol beta. These results identify several residues important for catalysis and quantitate the contributions of each of those residues. The functional data are discussed in relation to the prediction on the basis of available crystal structures.
Binding TS in preference to S and increasing TDeltaS++by freezing out motions in E X S and E X TS have been accepted as the driving forces in enzymatic catalysis; however, the smaller value of DeltaG++ for a one-substrate enzymatic reaction, as compared to its nonenzymatic counterpart, is generally the result of a smaller value of DeltaH++. Ground-state conformers (E X NACs) are formed in enzymatic reactions that structurally resemble E X TS. E X NACs are in thermal equilibrium with all other E X S conformers and are turnstiles through which substrate molecules must pass to arrive at the lowest-energy TS. TS in E X TS may or may not be bound tighter than NAC in E X NAC.
Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a "simplified" model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.
Understanding the chemical step in the catalytic reaction of DNA polymerases is essential for elucidating the molecular basis of the fidelity of DNA replication. The present work evaluates the free energy surface for the nucleotide transfer reaction of T7 polymerase by free energy perturbation/empirical valence bond (FEP/EVB) calculations. A key aspect of the enzyme simulation is a comparison of enzymatic free energy profiles with the corresponding reference reactions in water using the same computational methodology, thereby enabling a quantitative estimate for the free energy of the nucleotide insertion reaction. The reaction is driven by the FEP/EVB methodology between valence bond structures representing the reactant, pentacovalent intermediate, and the product states. This pathway corresponds to three microscopic chemical steps, deprotonation of the attacking group, a nucleophilic attack on the P(alpha) atom of the dNTP substrate, and departure of the leaving group. Three different mechanisms for the first microscopic step, the generation of the RO(-) nucleophile from the 3'-OH hydroxyl of the primer, are examined: (i) proton transfer to the bulk solvent, (ii) proton transfer to one of the ionic oxygens of the P(alpha) phosphate group, and (iii) proton transfer to the ionized Asp654 residue. The most favorable reaction mechanism in T7 pol is predicted to involve the proton transfer to Asp654. This finding sheds light on the long standing issue of the actual role of conserved aspartates. The structural preorganization that helps to catalyze the reaction is also considered and analyzed. The overall calculated mechanism consists of three subsequent steps with a similar activation free energy of about 12 kcal/mol. The similarity of the activation barriers of the three microscopic chemical steps indicates that the T7 polymerase may select against the incorrect dNTP substrate by raising any of these barriers. The relative height of these barriers comparing right and wrong dNTP substrates should therefore be a primary focus of future computational studies of the fidelity of DNA polymerases.
The recently determined structures of HIV-1 reverse transcriptase and Taq DNA polymerase in complex with DNA primer-template and an incoming nucleotide have shown that a large conformational change configures the polymerase active site for nucleotidyl transfer. The structure of reverse transcriptase in the catalytic complex will open the path to the rational design of novel nucleoside analog inhibitors of viral replication.
The kinetic mechanism and the structural bases of the fidelity of DNA polymerases are still highly controversial. Here we report the use of three probes in the stopped-flow studies of Pol beta to obtain new, direct evidence for our previous interpretations: (a) Increasing the viscosity of the reaction buffer by sucrose or glycerol is expected to slow down the conformational change differentially, and it was shown to slow down the first (fast) fluorescence transition selectively. (b) Use of dNTPalphaS in place of dNTP is expected to slow down the chemical step preferentially, and it was shown to slow down the second (slow) fluorescence transition selectively. (c) The substitution-inert Rh(III)dNTP was used to show for the first time that the slow fluorescence change occurs after mixing of Pol beta.DNA.Rh(III)dNTP with Mg(II). These results, along with crystal structures, suggest that the subdomain-closing conformational change occurs before binding of the catalytic Mg(II) while the rate-limiting step occurs after binding of the catalytic Mg(II). These results provide new evidence to the mechanism we suggested previously, but do not support the results of three recent papers of computational studies. The results were further supported by a "sequential mixing" stopped-flow experiment that used no analogues, and thus ruled out the possibility that the discrepancy between experimental and computational results is due to the use of analogues. The methodologies can be used to examine other DNA polymerases to answer whether the properties of Pol beta are exceptional or general.
Members of the ubiquitous family of peroxidases play an important role in protecting organisms from damage by reactive oxygen species such as hydrogen peroxide both in standard conditions and under oxidative stress. The family can be separated into three classes based on sequence similarity. Class I enzymes are the intracellular peroxidases of prokaryotic origin exemplified by yeast cytochrome c peroxidase that have a single covalently attached heme group. Class II enzymes are secreted fungal peroxidases, e.g., lignin peroxidase, and class III peroxidases are found in higher plants and include horseradish peroxidase. Classes II and III use small organic molecules as a source of electrons for the reaction, whereas the cytochrome c peroxidases (enzyme classification 1.11.1.5) in class I stand apart because they are very specific for their target, cytochrome c (Yonetani, 1976).
DNA polymerase (pol) β is a small eukaryotic DNA polymerase composed of two domains. Each domain contributes an enzymatic activity (DNA synthesis and deoxyribose phosphate lyase) during the repair of simple base lesions. These domains are referred to as the polymerase and lyase domains, respectively. Pol β has been an excellent model enzyme to study the nucleotidyl transferase reaction and substrate discrimination at a molecular level. In this review, recent crystallographic studies of pol β in various liganded and conformational states during the insertion of right and wrong nucleotides as well as during the bypass of damaged DNA (apurinic sites and 8-oxoguanine) are described. Structures of these catalytic intermediates provide unexpected insights into mechanisms by which DNA polymerases enhance genome stability. These structures also provide an improved framework that permits computational studies to facilitate the interpretation of detailed kinetic analyses of this model enzyme.
DNA polymerase β was the first mammalian DNA repair polymerase to be characterized. Although extensive studies have revealed this enzyme to display moderately high fidelity, a consensus has not yet been reached regarding the mechanism by which this is accomplished. This review considers structural and functional data for some of the recently discovered error-prone DNA polymerases including the African swine fever virus (ASFV) DNA polymerase X (Pol X). By comparison with higher fidelity enzymes such as Pol β, some conclusions are drawn about the mechanistic features contributing to relaxed polymerization fidelity. In the latter part of the review, fidelity is discussed as it pertains to DNA litigation. Finally, the complete ASFV-encoded DNA repair system, which displays low fidelity at both the DNA repair polymerization and nick ligation steps, is presented and the biological implications discussed.
The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3'-OH and catalytic Mg2+, resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here, we present a crystal structure of a precatalytic complex of a DNA polymerase with bound substrates that include the primer 3'-OH and catalytic Mg2+. This catalytic intermediate was trapped with a nonhydrolyzable deoxynucleotide analog. Comparison with two new structures of DNA polymerase beta lacking the 3'-OH or catalytic Mg2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3' on the alphaP of the incoming nucleotide.
A reference system for DNA replication fidelity was studied by free energy perturbation (FEP) and linear interaction energy (LIE) methods. The studied system included a hydrated duplex DNA with the 5'-CG dangling end of the templating strand, and dCTP4-.Mg2+ or dTTP4-.Mg2+ inserted opposite the dangling G to form a correct (i.e., Watson-Crick) or incorrect (i.e., wobble) base pair, respectively. The average distance between the 3'-terminal oxygen of the primer strand and the alpha-phosphorus of dNTP was found to be 0.2 A shorter for the correct base pair than for the incorrect base pair. Binding of the incorrect dNTP was found to be disfavored by 0.4 kcal/mol relative to the correct dNTP. We estimated that improved binding and more near-attack configurations sampled by the correct base pair should translate in aqueous solution and in the absence of DNA polymerase into a six times faster rate for the incorporation of the correct dNTP into DNA. The accuracy of the calculated binding free energy difference was verified by examining the relative free energy for melting duplex DNA containing GC and GT terminal base pairs flanked by a 5' dangling C. The calculated LIE and FEP free energies of 1.7 and 1.1 kcal/mol, respectively, compared favorably with the experimental estimate of 1.4 kcal/mol obtained using the nearest neighbor parameters. To decompose the calculated free energies into additive electrostatic and van der Waals contributions and to provide a set of rigorous theoretical data for the parametrization of the LIE method, we suggested a variant of the FEP approach, for which we coined a binding-relevant free energy (BRFE) acronym. BRFE approach is characterized by its unique perturbation pathway and by its exclusion of the intramolecular energy of a rigid part of the ligand from the total potential energy.
The control of the catalytic power and fidelity of DNA polymerases involves the complex combined effect of the protein residues, the Mg2+ ions, and the interaction between the DNA bases. In an attempt to advance the understanding of catalytic control, we analyze the effect of the protein residues, taking human DNA polymerase beta as a model system. Specifically, we examine the ability of different theoretical models to reproduce the effect of ionized residues on the transition state (TS) binding energy and the corresponding k(pol)/KD. We also explore the role of the Mg2+ ions in the binding and catalysis processes. The application of the microscopic linear response approximation (LRA) and the semimacroscopic PDLD/S-LRA methods to a benchmark of mutational studies produces a semiquantitative correlation and indicates that these methods can provide predictive power. However, pre-steady-state and steady-state kinetic studies currently available do not give a unique benchmark, owing principally to widely varying experimental conditions. We believe that a more uniform experimental benchmark is needed for further refinement of the theoretical models. The analysis of the correlation between the results obtained by a rigorous thermodynamic cycle and by simpler approximations indicates that the protein reorganization between the open, i.e., unbound, form and the closed form does not change the magnitude of the calculated mutational effects in a major way for the experimental data used in this study. The use of the PDLD/S-LRA group contributions allows us to construct energy-based correlation diagrams that can help toward understanding the coupling, i.e., transfer of information, between the base-binding and catalytic sites and to gain a deeper insight into the molecular basis of DNA replication fidelity. Our analysis suggests that the allosteric matrix obtained by subtracting the correlation matrix of the correct and incorrect base pairs should prove useful in exploring the information transfer occurring between the base-binding and catalytic sites. This type of treatment should be especially effective when coupled with structural studies of polymerase-DNA-base mispair ternary complexes and studies using polymerase double mutants. We discuss the potential of direct calculations of binding energy of the TS in a rational design of TS analogues and in drug design.
Free energy perturbation (FEP) calculations using the Amber 95 force field and the TIP3P water model were carried out to evaluate the solvation free energy of deoxyribonucleoside triphosphates in aqueous solution. Solvation free energies of -307.5, -311.5, -314.1, and -317.0 kcal/mol were calculated for the (Mg x dTTP)2-, (Mg x dATP)2-, (Mg x dCTP)2-, and (Mg x dGTP)2- complexes, respectively. Structural origins of the relative solvation free energies of deoxyribonucleoside phosphates were examined by calculating the contribution of the interaction of the base moiety with its surroundings. We showed that for each nucleobase the magnitude of this contribution is unaffected by substituting the 5'-OH group of the corresponding nucleoside with the charged mono- or triphosphate groups. This free energy contribution was further decomposed into the sum of free energies originating from the interactions of the base with itself, its substituent, water, and Na+ ions. Although the sum of these components was nearly constant over a wide range of solutes the individual free energy constituents varied significantly. Furthermore, this decomposition showed a high degree of additivity. Computational conditions necessary for obtaining additive free energy decomposition for the systems studied here within the framework of the FEP method included the use of a single mutation pathway and a subdivision of the FEP protocol into 51 or more windows.
Electrostatic energies provide what is perhaps the most effective tool for structure-function correlation of biological molecules. This review considers the current state of simulations of electrostatic energies in macromolecules as well as the early developments of this field. We focus on the relationship between microscopic and macroscopic models, considering the convergence problems of the microscopic models and the fact that the dielectric 'constants' in semimacroscopic models depend on the definition and the specific treatment. The advances and the challenges in the field are illustrated considering a wide range of functional properties including pK(a)'s, redox potentials, ion and proton channels, enzyme catalysis, ligand binding and protein stability. We conclude by pointing out that, despite the current problems and the significant misunderstandings in the field, there is an overall progress that should lead eventually to quantitative descriptions of electrostatic effects in proteins and thus to quantitative descriptions of the function of proteins.
With an increasing number of structural, kinetic, and modeling studies of diverse DNA polymerases in various contexts, a complex dynamical view of how atomic motions might define molecular "gates" or checkpoints that contribute to polymerase specificity and efficiency is emerging. Such atomic-level information can offer insights into rate-limiting conformational and chemical steps to help piece together mechanistic views of polymerases in action. With recent advances, modeling and dynamics simulations, subject to the well-appreciated limitations, can access transition states and transient intermediates along a reaction pathway, both conformational and chemical, and such information can help bridge the gap between experimentally determined equilibrium structures and mechanistic enzymology data. Focusing on DNA polymerase beta (pol beta), we present an emerging view of the geometric, energetic, and dynamic selection criteria governing insertion rate and fidelity mechanisms of DNA polymerases, as gleaned from various computational studies and based on the large body of existing kinetic and structural data. The landscape of nucleotide insertion for pol beta includes conformational changes, prechemistry, and chemistry "avenues", each with a unique deterministic or stochastic pathway that includes checkpoints for selective control of nucleotide insertion efficiency. For both correct and incorrect incoming nucleotides, pol beta's conformational rearrangements before chemistry include a cascade of slow and subtle side chain rearrangements, followed by active site adjustments to overcome higher chemical barriers, which include critical ion-polymerase geometries; this latter notion of a prechemistry avenue fits well with recent structural and NMR data. The chemical step involves an associative mechanism with several possibilities for the initial proton transfer and for the interaction among the active site residues and bridging water molecules. The conformational and chemical events and associated barriers define checkpoints that control enzymatic efficiency and fidelity. Understanding the nature of such active site rearrangements can facilitate interpretation of existing data and stimulate new experiments that aim to probe enzyme features that contribute to fidelity discrimination across various polymerases via such geometric, dynamic, and energetic selection criteria.
DNA polymerase catalysis and fidelity studies typically compare incorporation of "right" versus "wrong" nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the beta,gamma-bridging O with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) beta. T.G mismatches occur with fidelities similar to dGTP with the exception of the CH2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with Kds between 1 and 4 microM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brnsted correlations of log(kpol) versus leaving-group pKa for both right and wrong base incorporation reveal similar sensitivities (betalg approximately -0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pKas of CF2, O, and the sterically bulky CCl2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3'-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.
Structure and mechanism of DNA polymerase β
- W A Beard
- S H Wilson
Beard WA, Wilson SH. Structure and mechanism of DNA polymerase β. Chem Rev 2006;106:361-82. [PubMed: 16464010]
Magnesium-Induced Assembly of a DNA Polymerase Catalytic Complex
- V K Batra
- W A Beard
- D D Shock
- J M Krahn
- L C Pedersen
- S H Wilson
Batra VK, Beard WA, Shock DD, Krahn JM, Pedersen LC, Wilson SH. Magnesium-Induced
Assembly of a DNA Polymerase Catalytic Complex. Structure 2006;14:1-10. [PubMed: 16407058]