Computational delineation of the catalytic step of a high-fidelity DNA polymerase

Abstract

The Bacillus fragment, belonging to a class of high-fidelity polymerases, demonstrates high processivity (adding approximately 115 bases per DNA binding event) and exceptional accuracy (1 error in 10(6) nucleotide incorporations) during DNA replication. We present analysis of structural rearrangements and energetics just before and during the chemical step (phosphodiester bond formation) using a combination of classical molecular dynamics, mixed quantum mechanics molecular mechanics simulations, and free energy computations. We find that the reaction is associative, proceeding via the two-metal-ion mechanism, and requiring the proton on the terminal primer O3' to transfer to the pyrophosphate tail of the incoming nucleotide before the formation of the pentacovalent transition state. Different protonation states for key active site residues direct the system to alternative pathways of catalysis and we estimate a free energy barrier of approximately 12 kcal/mol for the chemical step. We propose that the protonation of a highly conserved catalytic aspartic acid residue is essential for the high processivity demonstrated by the enzyme and suggest that global motions could be part of the reaction free energy landscape.

Figure 1

The bacillus fragment (BF) complexed with its DNA substrate and incoming deoxynucleotide triphosphate (dNTP); we study the incorporation of dCTP opposite a guanine (G) template base. The inset shows a magnified view of the catalytic site. The phosphoryl transfer reaction involves the formation of a covalent bond (solid line) between the α-phosphorous (P_α) of the incoming dNTP and O3′ oxygen of the terminal primer base, before which a proton present on O3′ has to transfer (dashed line) possibly to a highly conserved aspartic acid residue. [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com.]

Figure 2

Schematics of the reaction pathway for catalysis (a) and (c). An effective 1-d free energy landscape along a generalized coordinate defined along the minimum free energy path (b) and (d) for two different models: model III (top row) and model I (bottom row). In (a) and (c), the newly forming O3′-P_α bond and cleaving P_α-O_3α bond are illustrated by solid and dashed black lines, respectively. The other arrows indicate different proton transfer steps. [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com.]

Figure 3

1-d and 2-d free energy surfaces for model III obtained from umbrella sampling simulations. The surfaces are labeled in chronological order (a)–(d) as the reaction proceeds from reactant to product. [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com.]

Figure 4

1-d and 2-d free energy surfaces for model I obtained from umbrella sampling simulations. The surfaces are labeled in chronological order (a)–(d) as the reaction proceeds from reactant to product. Error bars, when not visible, are smaller than the size of the symbols. [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com.]

Figure 5

(a) A comparison of free energy change with displacement a₁ along principal component mode ξ₁ as obtained from umbrella sampling simulations (blue) and using the spring constant K₁ resulting from quasiharmonic analysis (black). (b) Free energy surface (ΔU_UMB in units of k_BT) obtained from classical umbrella sampling runs using the O3′-P_α and O3′-catalytic Mg²⁺ distances as reaction coordinates. The lines of filled green, yellow, and red squares represent the change in the two reaction coordinates for a displacement along the top three PC modes ξ₁,ξ₂, and ξ₃, respectively. The reference geometry is the minimum free energy state (white square marked 1.5 k_BT) and each mode is displaced by amplitudes yielding the maximum simultaneous reduction of both reaction coordinates (white squares marked 5.1 k_BT, 3.4 k_BT and 6.1 k_BT). (c) Free energy change ΔU_PC with displacement a_n (n = 1, 2, 3) along the top three PC modes ξ₁,ξ_2, and ξ₃ as obtained using their respective spring constants K₁, K_2^, and K₃ (resulting from quasiharmonic analysis). (d) comparison of free energy change ΔU_UMB to ΔU_PC for achieving the same reduction in distances d_a and d_b.