Quantum mechanics/molecular mechanics investigation of the chemical reaction in Dpo4 reveals water-dependent pathways and requirements for active site reorganization

Abstract

The nucleotidyl-transfer reaction coupled with the conformational transitions in DNA polymerases is critical for maintaining the fidelity and efficiency of DNA synthesis. We examine here the possible reaction pathways of a Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4), for the correct insertion of dCTP opposite 8-oxoguanine using the quantum mechanics/molecular mechanics (QM/MM) approach, both from a chemistry-competent state and a crystal closed state. The latter examination is important for understanding pre-chemistry barriers to interpret the entire enzyme mechanism, since the crystal closed state is not an ideal state for initiating the chemical reaction. The most favorable reaction path involves initial deprotonation of O3'H via two bridging water molecules to O1A, overcoming an overall potential energy barrier of approximately 20.0 kcal/mol. The proton on O1A-P(alpha) then migrates to the gamma-phosphate oxygen of the incoming nucleotide as O3' attacks P(alpha), and the P(alpha)-O3A bond breaks. The other possible pathway in which the O3'H proton is transferred directly to O1A on P(alpha) has an overall energy barrier of 25.0 kcal/mol. In both reaction paths, the rate-limiting step is the initial deprotonation, and the trigonal-bipyramidal configuration for P(alpha) occurs during the concerted bond formation (O3'-P(alpha)) and breaking (P(alpha)-O3A), indicating the associative nature of the chemical reaction. In contrast, the Dpo4/DNA complex with an imperfect active-site geometry corresponding to the crystal state must overcome a much higher activation energy barrier (29.0 kcal/mol) to achieve a tightly organized site due to hindered O3'H deprotonation stemming from larger distances and distorted conformation of the proton acceptors. This significant difference demonstrates that the pre-chemistry reorganization in Dpo4 costs approximately 4.0 to 9.0 kcal/mol depending on the primer terminus environment. Compared to the higher fidelity DNA polymerase beta from the X-family, Dpo4 has a higher chemical reaction barrier (20.0 vs 15.0 kcal/mol) due to the more solvent-exposed active site.

Figure 1

The molecular formulas of guanine (a) and 8-oxoguanine (b).

Figure 2

(a) Initial simulation model of the solvated Dpo4/DNA/8-oxoG:dCTP complex. (b) Reduced model of the Dpo4/DNA/8-oxoG:dCTP complex. All atoms within 15 Å of any QM atom are freely optimized during the minimizations, except for the restraints imposed to follow the reaction pathway. Atoms between 15 and 20 Å of any QM atom are semi-constrained and atoms beyond 20 Å are fixed in their original positions. In both models, the Dpo4 protein and its binding DNA are shown in red cartoon representation; the sodium, chloride, and magnesium ions are rendered as yellow, light blue and green spheres, respectively; the incoming dCTP is shown by bonds, and solvent water molecules are shown by grey lines.

Figure 3

The catalytic sites of Dpo4 in the optimized (a) and the crystal state models (b). QM atoms are enclosed in the black circles. The missing 3′OH of the primer terminus was added using CHARMM in both models. The critical distances, namely O3′ – Mg²⁺ (cat.), Mg²⁺ (cat.)–O2A, and P_α–O3′ (in Å) are shown by dashed lines and labeled in red.

Figure 4

Intermediates along the water-assisted deprotonation pathway, where two explicit water molecules abstract the O3′H proton and transfer it to O1A of the incoming dCTP. (a) Initial reactant state (the two water molecules involved in abstracting the O3′H proton are circled with dashed blue lines); (b) intermediate I, proton on O1A; (c) intermediate II, proton rotation around O1A-P_α; (d) intermediate III, pentacovalent P_α; (e) product state. Scheme 1 is a schematic representation of the water-assisted deprotonation pathway in the optimized system. Arrows show the direction of proton transfer from O3′-hydroxyl through the mediating water molecules (WAT5, WAT73, and WAT2) to the leaving pyrophosphate, as well as the direction of nucleophilic attack by O3′ at the α-phosphate.

Figure 5

The energy profile for the transient intermediates identified in the water-assisted deprotonation reaction pathway (labeled corresponding to Figure 4).

Figure 6

Intermediates along the phosphate-oxygen-assisted reaction pathway, where O1A of the incoming dCTP functions as the general base to abstract the O3′H proton. (a) Reactant state; (b) intermediate I, proton on O1A; (c) intermediate II, proton rotation around O1A-P_α; (d) intermediate III with a pentacovalent P_α; (e) product state. A schematic representation of the phosphate-oxygen-assisted deprotonation pathway in the optimized system is shown in Scheme 2. Arrows indicate the direction of proton transfer from O3′-hydroxyl directly to O1A and through WAT2 to the leaving pyrophosphate, as well as the direction of nucleophilic attack by O3′ at the α-phosphate.

Figure 7

The energy profile for the transient intermediates identified in the phosphate-oxygen-assisted reaction pathway (labeled as in Figure 6).

Figure 8

Intermediates along the reaction pathway in the crystal model where proton abstraction occurs directly to O1A of the incoming dCTP. (a) Reactant state after removing the two water molecules from the active site (the critical distances (Å) of Mg²⁺ (cat.)–O2A, Mg²⁺ (cat.)–O3′, and O3′–P_α are shown by dashed lines and labeled in red); (b) intermediate I, proton on O1A; (c) intermediate II, proton rotation around O1A-P_α; (d) product state. The overall phosphate-oxygen-assisted deprotonation pathway in the crystal system with imperfect active-site geometry is also depicted by schematic drawing in Scheme 3. Arrows show the direction of proton transfer from O3′-hydroxyl directly to O1A and that of nucleophilic attack by O3′ at the α-phosphate.

Figure 9

The energy profile for the transient intermediates identified in the phosphate-oxygen-assisted reaction pathway of the crystal system (labeled as in Figure 8).