Structural insights into complete metal ion coordination from ternary complexes of B family RB69 DNA polymerase

Abstract

We have captured a preinsertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,β-substituted triphosphate, dUpXpp, where X is either NH or CH(2), opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca(2+) and four other structures with catalytically competent Mn(2+) or Mg(2+). These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3' close to the α-phosphorus atom (Pα) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the nonbridging Sp oxygen of the dNTP's α-phosphate. The coordination bond length of metal ion A as well as its ionic radius determines how close ptO3' can approach Pα. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to Pα to enable pyrophosphorolysis and assist in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3' occupies the vertex of a distorted metal ion A coordination octahedron. When ptO3' is placed at the vertex of an undistorted, idealized metal ion A octahedron, it is within bond formation distance to Pα. This geometric relationship appears to be conserved among DNA polymerases of known structure.

Figure 1

Ca²⁺ coordination in the dUpNpp-containing ternary complexes. (A) The wt complex in the presence of a 2’,3’-dideoxy-terminated primer (ddC). (B) The Ca²⁺ coordination in the tm complex (golden) is exactly the same as in the wt complex (multi-colors as in A). (C) The tm complex in the presence of an extendable primer (dC). (D) Coordination comparison of the ternary complexes in the presence of dC (yellow) and ddC (golden) shows that the removal of ptO3’ in ddC is responsible for introduction of more than 6 ligands for Ca²⁺ coordination, for example, for the addition of W-4 in (A). (E) The dCTP-containing ternary complex of wt RB69pol published previously (pdb entry, 3NCI) shows a new water molecule W-7 coordinated to CaA, but missing the bridging water molecules W-4. (F) Coordination comparison of the dCTP (silver) and dUpNpp-containing (multi-colors) wt ternary complexes shows altered water molecule ligands, where W-4 is part of hydration network connecting to the α,β -bridging NH of dUpNpp.

Figure 2

Mn²⁺ coordination in the dUpNpp-containing ternary complex. (A) Anomalous difference Fourier maps contoured at 6.5 σ (red) and the omitted Fobs-Fcalc maps contoured at 1 σ (cyan) in stereodiagram show well defined Mn²⁺ coordination. (B) Mn²⁺ coordination details: B site, nearly a perfect octahedron; A site, a distorted octahedron by ptO3’ (C) Coordination comparison of the ternary complexes in the presence of Mn²⁺ (multi-colors) and Ca²⁺ (silver) shows that the smaller ionic radius of Mn²⁺ than Ca²⁺ causes a contraction in both A and B sites, adding a stronger interaction between ptO3’ and MnA than between ptO3’ and CaA. (D) Details of Mn²⁺ coordination in stereodiagram with outlined octahedrons. (E) The Mn²⁺/dUpNpp-containing tm ternary complex in the absence of ptO3’ (F) Comparison of the Mn²⁺/dUpNpp-containing tm ternary complexes in the presence (silver) and absence of ptO3’ (mutli-color as in E).

Figure 3

Mg²⁺ coordination in the dUpCpp-containing qm ternary complex. (A) Complete coordination of the Mg²⁺/dUpCpp-containing qm ternary complex. (B) Comparison of the Mg²⁺/dUpCpp-containing qm ternary complex (multicolor) with the Mn²⁺/dUpNpp-containing tm ternary complex (silver). (C) Stereodiagram of the Mg²⁺ coordination.

Figure 4

The Ca²⁺/dUpCpp qm ternary complexes. (A) The Ca²⁺/dUpCpp-containing qm ternary complex. (B-C) Two orthogonal views of comparison of the Ca²⁺/dUpCpp-containing qm ternary complex (multicolor) with the Ca²⁺/dUpNpp-containing tm ternary complex (silver). (D) Comparison of the Ca²⁺/dUpCpp-containing qm ternary complex (multicolor) with the Ca²⁺/dUpNpp-containing wt ternary complex (silver). (E-F) Two orthogonal views of comparison of Ca²⁺/dUpCpp-containing qm ternary complex (multicolor) with the Mg²⁺/dUpCpp-containing qm ternary complex (silver).

Figure 5

Mechanistic implications from the α,β,γ-tridendate Mg²⁺/Mn²⁺ coordination complex. (A) A displacement of ptO3’ into the vertex of a non-distorted, idealized metal-ion A octahedron (as indicated in black arrows and octahedron outlines) would place it in a distance for bond formation with Pα and in a hypothetic TS model. Pα is also displaced towards ptO3’ for the hypothetic Pα-center inversion (see text). (B) Two idealized metal-ion coordination octahedrons linked by shared ligands. (C) Geometric relationship between the two idealized metal-ion coordination octahedrons (cyan and yellow) and a hypothetic pentavalent TS (magenta). (D) Coordination comparison of the dUpNpp-containing ternary complexes of RB69pol (golden) and pol β (yellow) upon superposition using metal ion ligands. Second residue labels are for corresponding catalytic carboxylates of pol β. (E) Coordination comparison of the dUpNpp-containing ternary complex of RB69pol (yellow) with dNTP-containing ternary complexes of other pols: T7 DNA polymerase (green), HIV reverse transcriptase (cyan), and Dpo4 (magenta). Three B site ligands are backbone carbonyl (Z, L412, A476, V111, and F8, respectively, for the above-mentioned four pols) and two carboxylates (DX/DY, D411/D623, D475/D654, D110/D185, and D7/D105).