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, 366 (2), 687-701

Magnesium-cationic Dummy Atom Molecules Enhance Representation of DNA Polymerase Beta in Molecular Dynamics Simulations: Improved Accuracy in Studies of Structural Features and Mutational Effects

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Magnesium-cationic Dummy Atom Molecules Enhance Representation of DNA Polymerase Beta in Molecular Dynamics Simulations: Improved Accuracy in Studies of Structural Features and Mutational Effects

Peter Oelschlaeger et al. J Mol Biol.

Abstract

Human DNA polymerase beta (pol beta) fills gaps in DNA as part of base excision DNA repair. Due to its small size it is a convenient model enzyme for other DNA polymerases. Its active site contains two Mg(2+) ions, of which one binds an incoming dNTP and one catalyzes its condensation with the DNA primer strand. Simulating such binuclear metalloenzymes accurately but computationally efficiently is a challenging task. Here, we present a magnesium-cationic dummy atom approach that can easily be implemented in molecular mechanical force fields such as the ENZYMIX or the AMBER force fields. All properties investigated here, namely, structure and energetics of both Michaelis complexes and transition state (TS) complexes were represented more accurately using the magnesium-cationic dummy atom model than using the traditional one-atom representation for Mg(2+) ions. The improved agreement between calculated free energies of binding of TS models to different pol beta variants and the experimentally determined activation free energies indicates that this model will be useful in studying mutational effects on catalytic efficiency and fidelity of DNA polymerases. The model should also have broad applicability to the modeling of other magnesium-containing proteins.

Figures

Figure 1
Figure 1
Representation of the active sites of (A) the MgMg structure and (B) the MgNa structure used for MD simulations. The imido nitrogen bridging the α- and β-phosphate groups of the dUTP analogue in the crystal structures was mutated to a phosphodiester oxygen and the uracil part was mutated to thymidine, thus generating dTTP. The side chains and Cα atoms of amino acids are shown as sticks, colored by atom (cyan, C; blue, N; red, O), and labeled at the Cα atom. Nucleotides are shown as sticks, colored by atom (copper, P), and labeled at the base. Oxygens of water molecules are shown as red spheres. Mg2+ions are shown as grey spheres and the Na+ ion as a blue sphere. (A) The two Mg2+ ions Mg(b) and Mg(c) and their ligands are shown. Both metal ions are coordinated octahedrally. In addition, the templating nucleotide (template A) and amino acids that were mutated in this study (Arg149, Arg183, and Lys280) are shown. (B) The Mg2+ ion Mg(b) and the Na+ ion Na(c) and their ligands are shown. While Mg(b) is coordinated octahedrally, Na(c) is coordinated in a distorted tetrahedral fashion with relatively large Na(c)-ligand distances (2.19–2.74 Å). Note that the water molecule coordinated to Mg(c) [WAT(c)] in the MgMg structure is missing in the MgNa structure and that O3′ of the primer C is not ligating Na(c) (3.54 Å) and is not positioned for nucleophilic attack on the dTTP αP (4.70 Å). The figures were generated with VMD, version 1.8.3.
Figure 1
Figure 1
Representation of the active sites of (A) the MgMg structure and (B) the MgNa structure used for MD simulations. The imido nitrogen bridging the α- and β-phosphate groups of the dUTP analogue in the crystal structures was mutated to a phosphodiester oxygen and the uracil part was mutated to thymidine, thus generating dTTP. The side chains and Cα atoms of amino acids are shown as sticks, colored by atom (cyan, C; blue, N; red, O), and labeled at the Cα atom. Nucleotides are shown as sticks, colored by atom (copper, P), and labeled at the base. Oxygens of water molecules are shown as red spheres. Mg2+ions are shown as grey spheres and the Na+ ion as a blue sphere. (A) The two Mg2+ ions Mg(b) and Mg(c) and their ligands are shown. Both metal ions are coordinated octahedrally. In addition, the templating nucleotide (template A) and amino acids that were mutated in this study (Arg149, Arg183, and Lys280) are shown. (B) The Mg2+ ion Mg(b) and the Na+ ion Na(c) and their ligands are shown. While Mg(b) is coordinated octahedrally, Na(c) is coordinated in a distorted tetrahedral fashion with relatively large Na(c)-ligand distances (2.19–2.74 Å). Note that the water molecule coordinated to Mg(c) [WAT(c)] in the MgMg structure is missing in the MgNa structure and that O3′ of the primer C is not ligating Na(c) (3.54 Å) and is not positioned for nucleophilic attack on the dTTP αP (4.70 Å). The figures were generated with VMD, version 1.8.3.
Figure 2
Figure 2
Scheme of the model complexes used for the calculations presented in Tables III and IV. The measured distances are the distances between the central atom M and the ligand X of interest in the intact complex shown on the left. Dissociation energies were calculated as the difference between the energy of the intact complex [MD6(H2O)5]2+X (left side) and the sum of the energies of the fragments [MD6(H2O)5]2+ and X resulting from dissociation (right side). For DFT calculations, the MD62+ molecule was replaced by a Mg2+ atom.
Figure 3
Figure 3
Critical distances in the active site of the MgMg structure over the course of 1 ns MD simulations at different simulation temperatures (panels A, B, and C: 100 K; panels D, E, and F: 200 K; panels G, H, and I: 300 K) using the regular Mg2+ model (black) and the MD62+ model (grey). Panels A, D, and G show the Mg(b)-Mg(c) distance; panels B, E, and H the Mg(b)-O1A distance; and panels C, F, and I the Mg(b)-O2A distance.
Figure 4
Figure 4
Critical distances in the active site of the MgNa structure over the course of 1 ns MD simulations at different simulation temperatures (panels A, B, and C: 100 K; panels D, E, and F: 200 K; panels G, H, and I: 300 K) using the regular Mg2+ model (black) and the MD62+ model (grey). Panels A, D, and G show the Mg(b)-Na(c) distance; panels B, E, and H the Mg(b)-O1A distance; and panels C, F, and I the Mg(b)-O2A distance.
Figure 5
Figure 5
The experimentally determined pre-steady-state substrate binding energies ΔGbind, exp. are plotted versus the calculated substrate binding free energies ΔGbind, calc., which were obtained using the regular Mg2+ model (panel A) or the MD62+ model (panel B). The following pol β variants/nascent base pairs (template base:incorporated base) are shown: ■ WT/A:T; ● R149A/A:T; ◆ R183A/A:T; □WT/G:C; ○ R149A/G:C; ◇ R183A/G:C; △ K280A/G:C.
Figure 6
Figure 6
The experimentally determined equilibrium TS binding energies Δgenz, exp. are plotted versus the calculated TS binding free energies Δgenz, calc., which were obtained using the regular Mg2+ model (panel A) or the MD62+ model (panel B). The following pol β variants/nascent base pairs (template base:incorporated base) are shown: ■ wild type (WT)/A:T; ● R149A/A:T; ♦ R183A/A:T; □ WT/G:C; ○ R149A/G:C; ⋄R183A/G:C; △ K280A/G:C.

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