Revealing the role of the product metal in DNA polymerase β catalysis

Abstract

DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase β, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction.

Figure 1.

Active site structure of the ternary complexes of pol β. (A) Reactant state from crystallographic structure (PDB ID: 4KLE) having magnesium ions in the catalytic and nucleotide binding sites; (B) product state from crystallographic structure (PDB ID: 4KLG) having magnesium ions in the nucleotide and product metal sites and a sodium ion in the catalytic metal site; (C) reactant state from the final point of a lengthy well equilibrated MD trajectory calculation based on the structure in panel (A) where the product metal ion was initially introduced to the system based on the location of this metal in a crystallographic product structure (PDB ID: 4KLG). Important distances (Å) are indicated (red dashed lines) as are metal coordination (black dashed lines). Two water molecules solvating the product metal make hydrogen bonds with phosphate oxygens of the primer terminal nucleotide and Pγ of the incoming dCTP (green dashed lines).

Figure 2.

Energy profiles for the pol β nucleotidyl transfer reaction. (A) Profile obtained from QM/MM calculations with either two (blue) or three (red) magnesium ions. The reaction coordinate is chosen to be the distance between the primer terminal nucleophile O3΄ and Pα of the incoming dCTP; the distance is reduced from right to left. In the designated transition state region (TS), the dissociating Pα–Oαβ bond has been stretched by 0.2 Å over its equilibrium distance. Also, the reactant (RS) and product states (PS) are indicated. (B) The starting (gray) and final (color) configurations of the QM sub-system (100 atoms) in the three magnesium calculation. Magnesium ions are represented by solid spheres (gray or green). All quantum waters are shown. Key water and Oαβ interactions in the final state are indicated with red dashed lines. (C) The starting and final configurations of the QM sub-system (99 atoms) with two magnesium ions. A water interaction with Oαβ in the final state is highlighted (red dashed line).

Figure 3.

Crystallographic structure of a product complex formed after insertion of the S_p-isomer of dCTPαS. (A) Active site after insertion of the S_p-isomer of dCTPαS. An anomalous density map contoured to 5σ is shown indicating that Mn²⁺ occupies the catalytic and nucleotide metal binding sites. (B) Pol β active site structural comparison of the products of dCTP (PDB ID 4KLH) and S_p-dCTPαS (PDB ID 5U9H) insertion. A product metal is observed only during the incorporation of a natural nucleotide. Sulfur (transparent yellow) appears to exclude product metal binding. The equivalent oxygen of dCTP (i.e. pro-S_p) is red (appears orange in the overlap with the sulfur van der Waals radius). Three Mn²⁺ (gray) are observed during insertion of dCTP whereas only two manganese ions (purple) are detected during the insertion of the S_p-isomer of dCTPαS.

Figure 4.

Energy profiles for the pol β nucleotidyl transfer reaction. (A) Profile obtained from QM/MM calculations with a sodium ion in the product metal site (green). The two- (blue) and three-metal (red) site profiles are also shown for comparison (see Figure 2). This system is used to account for an ion near the product metal binding site that could potentially exhibit the transient behavior during catalysis. The transition (TS, described in the text), reactant (RS), and product states (PS) are indicated. (B) The starting (gray) and final (color) QM sub-system configurations (100 atoms) of the three-metal system with a sodium ion at the product metal site are shown. Metal ions are represented by solid spheres (gray or green). The red dashed line represents a newly formed interaction between a Na(p)-bound water and Oαβ in the final state.

Figure 5.

Metal coordination distance variations along the reaction coordinate. (A) Coordination distances for the catalytic, (B) nucleotide binding, and (C) product metal sites. (D) Metal–metal distances along the reaction coordinates (top panel, Mg(c)–(Mg(n) for all cases; bottom panel, Mg(c)–M(p) and Mg(n)–M(p) with M being magnesium or sodium). In (A) and (B), the Mg(c)Mg(n), Mg(p) system is in the top panel, the Mg(c)Mg(n) system is in the middle panel, and the Mg(c)Mg(p)Na(p) system is in the bottom panel. The same color scheme for the top panel is followed for the other panels in (A–C).

Figure 6.

Hydrogen-bond network that connects the phosphate group on the primer terminus with the dNTP (or PP_i). Involvement of the product metal in strengthening the network is apparent. (A) Mg(c)Mg(n)Mg(p), (B) Mg(c)Mg(n), and (C) Mg(c)Mg(n)Na(p). Hydrogen bonds are indicated in purple dashed lines and the product metal coordination is shown in blue.

Figure 7.

Energy profiles for the pol β nucleotidyl transfer reaction. The profile (black) calculated with an ammonium ion is used to mimic a charged lysine residue found in the position of Mg(p) (13). This lysine residue is conserved in A- and B-family DNA polymerases. The energy profiles of the other three systems are included to facilitate comparison.