Energy analysis of chemistry for correct insertion by DNA polymerase ␤ Ping Lin†, Lars C. Pedersen‡, Vinod K. Batra‡, William A. Beard‡, Samuel H. Wilson‡, and Lee G. Pedersen†‡§ †Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599; and ‡Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709-2233 Communicated by Robert G. Parr, University of North Carolina, Chapel Hill, NC, July 19, 2006 (received for review March 8, 2006) X-ray crystallographic structures of human DNA polymerase ␤ with transition-state (TS) theory applied to an enzyme-catalyzed nonhydrolyzable analogs containing all atoms in the active site reaction (27) and an insertion rate constant of 10 sϪ1 measured required for catalysis provide a secure starting point for a theo- for gapped DNA (28), the apparent free energy of activation is retical analysis (quantum mechanics͞molecular mechanics) of the estimated to be 16.1 kcal (1 kcal ϭ 4.18 kJ)͞mol at room mechanism of chemistry without biasing of modeling assumptions temperature. Although there is some debate about the extent to as required in previous studies. These structures provide the basis which a conformational change or the chemical reaction is the for a detailed quantum mechanics͞molecular mechanics study of rate-limiting step (29), we may use this value for comparison with the path for the complete transfer of a monophosphate nucleoside our calculated reaction energy barrier. donor to the sugar acceptor in the active site. The reaction is largely The development of combined quantum mechanics (QM) and associative with the main energetic step preceded by proton molecular mechanics (MM) methods provides tools for investi- transfer from the terminal primer deoxyribose O3؅ to Asp-256. The gating the catalytic process for complex biological systems. The key residues that provide electrostatic stabilization of the transi- reactive center, where bonds break and form, is treated with an tion state are identified and compared with those identified by accurate QM approach, whereas the remainder of the enzyme mutational studies. environment is treated with empirical MM (30–32). There have been a number of successful applications of the QM͞MM quantum mechanics ͉ molecular mechanics ͉ reaction mechanism method in the studies of the mechanism of enzyme-catalyzed chemical reactions (refs. 33 and 34 and references therein). In ͞ NA polymerases play a central role in the replication and this study, the QM MM version of the ONIOM method devel- oped by the Morokuma group (35, 36) is used, with the electronic repair of DNA. High-fidelity proofreading-deficient poly- D coupling between the QM and MM regions implemented merases typically produce one error per million nucleotides through an electronic embedding approach. synthesized (1). DNA polymerase ␤ (pol ␤) is the simplest eukaryotic DNA polymerase and catalyzes the template- Results and Discussion directed nucleotidyl transfer reaction during the repair of ‘‘sim- We used a constrained dynamics simulation͞no-constraint min- ple’’ base lesions with moderate fidelity, producing approxi- imization procedure, which resulted in a full-atom model that mately one error per 3,000 nt synthesized during base excision well represents the initial structure before the reaction. The DNA repair (2). DNA pol ␤ has been characterized by x-ray rmsd of the backbone atoms between the minimized structure crystallography in various liganded states (3–8), and the dynam- and the crystal structure was 0.31 Å. The unconstrained mini- ics were characterized by NMR (9, 10) and time-resolved mized structure with the Amber ff99 force field was further fluorescence (11). A number of theoretical studies have been ͞ ␤ optimized at the ONIOM(B3LYP 3–21G*:Amber) and carried out with pol in an effort to understand details of ONIOM(B3LYP͞6–31G*:Amber) levels. A comparison of key catalysis (12–15). Additionally, computer modeling of confor- active-site distances between optimized structures and crystal mational changes deduced from x-ray crystallography before and structure is presented in Fig. 3, which is published as supporting after ligand binding and catalysis have provided insight into information on the PNAS web site. Overall, we found that the substrate discrimination (16–23). These structural and compu- geometry of the active site optimized by the QM͞MM hybrid tational studies can be interpreted in the wealth of kinetic and potential agrees well with the experimental values and signifi- ␤ site-directed mutagenesis studies available for pol (2). cantly better than if optimized with the classical force field. One A recent structural study of the precatalytic complex including of the most notable differences is in the coordination between ␤ Ј pol , DNA template, primer and dNTP analogue, 2 -deoxy- the Mg2ϩ (binding) and the O1A of the ␣-phosphate, which Ј ␣ ␤ uridine-5 -( , )-imido triphosphate, provides a ternary complex changes from a noncoordination distance at 2.79 Å (classical) to Ј structure including a hydroxyl group (O3 ) on the primer a distance at Ϸ2.1 Å (QM͞MM). The positions of some sur- terminus with two bound Mg ions (24). Both Mg ions are found rounding x-ray crystallographic waters and ions also changed to be octahedrally coordinated, with each ion bound firmly to a after simulation. ␣ nonbridging oxygen on the dNTP -phosphate and each with a We also investigated the interactions between the critical 3Ј distinct bound water molecule. The primer O3Ј atom, often hydroxyl group of the primer terminus and the surrounding missing in other x-ray crystal structures and necessary for the environment. The optimized structure with the Amber99 force nucleophilic attack, is securely bound in the inner coordination field suggests a H-bonding interaction involving the hydrogen sphere of the catalytic Mg ion. As all of the vital components for (H3T) of the terminal 3Ј hydroxyl group and the O5Ј on the the reaction are in place in this experimental structure, we have incoming nucleotide triphosphate, an interaction also suggested a secure initial reaction system that minimizes the chance of in an earlier study (15). However, such an initial configuration introducing modeling bias at the outset of a theoretical study. All families of DNA polymerases are believed to catalyze the nucleotidyl transfer reaction through a universal two-metal-ion Conflict of interest statement: No conflicts declared. mechanism (25). Therefore, the detailed reaction pathway we Abbreviations: pol ␤, polymerase ␤; QM, quantum mechanics; MM, molecular mechanics; may derive from this study should provide a general picture of TS, transition state. nucleotidyl transfer reactions. The nucleotide insertion reaction §To whom correspondence should be addressed. E-mail: [email protected]. catalyzed by pol ␤ occurs at a moderate rate (26). Based on © 2006 by The National Academy of Sciences of the USA 13294–13299 ͉ PNAS ͉ September 5, 2006 ͉ vol. 103 ͉ no. 36 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606006103 Downloaded by guest on October 1, 2021 of H3T does not provide a viable pathway for the critical deprotonation of the 3Ј hydroxyl group. Exploring the potential energy surface with the QM͞MM potential allows us to identify a second energy minimum in which H3T forms a H bond with OD2 of Asp-256. This minimum is Ϸ4 kcal͞mol lower in energy than the energy minimum of the structure with the H3T- O5Ј(dTTP) H bond. This configuration for H3T suggests a direct proton transfer pathway from the 3Ј hydroxyl primer terminus to Asp-256. Proton transfer to an ␣-phosphate oxygen suggested by a cluster model calculation (12) appears unlikely because such a proton transfer pathway will require large structural rearrange- ment of the active site, which is hindered by the surrounding residues in the initial structure. A water-mediated proton trans- fer pathway is also possible if water molecules are able to penetrate and interact with H3T. The proton will subsequently be transferred to the OD1 of Asp-256 (unpublished result) or migrate to the solution (37, 38). Reaction Mechanism. The deprotonation of the terminal hydroxyl group of the growing polynucleotide primer is believed to be an essential initial step for the success of the nucleotidyl transfer reaction. The proton transfer occurred automatically before the formation of O3Ј-P␣ bond when we drove the reaction forward by reducing the O3Ј-P␣ distance. A reverse exploration of the potential energy surface identifies a local minimum of the proton transfer product with an energy 3.5 kcal͞mol higher than the reactant state. The existence of the local minimum of the proton transfer product CHEMISTRY suggests the reaction proceeds by a two-step pathway, in which proton transfer reaction occurs before the nucleotidyl transfer reaction. Because the proton transfer reaction proceeds with a relatively lower energy barrier, the overall activation barrier is determined by the formation of O3Ј-P1A bond and the breaking of P1A-O3A bond. The rate-determining process in such a two-step reaction will have a lower activation barrier than the single-step process and therefore improves the catalytic proficiency. An illus- tration of the potential energy profile is presented in Fig. 4, which BIOCHEMISTRY is published as supporting information on the PNAS web site. The key active-site structures along the reaction path have been ob- tained in our calculations and are illustrated in Fig. 1. The transfer Fig. 1. Critical structures in the QM region determined along the reaction of the proton to Asp-256 significantly weakens the side-chain pathway. (a) Structures of ground state, local minimum state, TS, and product coordination with Mg2ϩ(catalytic) and enhances the ligand-metal state. Blue indicates the sugar of the primer terminus, green indicates Mg ions, interaction between O3Ј and Mg2ϩ(catalytic). The distance be- and yellow indicates phosphates. A nonmetal coordinated water molecule Ј 2ϩ included in the quantum atoms is deleted for clarity.