Mapping Functional Substrate-Enzyme Interactions in the pol β Active Site through Chemical Biology: Structural Responses to Acidity Modification of Incoming dNTPs

Abstract

We report high-resolution crystal structures of DNA polymerase (pol) β in ternary complex with a panel of incoming dNTPs carrying acidity-modified 5'-triphosphate groups. These novel dNTP analogues have a variety of halomethylene substitutions replacing the bridging oxygen between Pβ and Pγ of the incoming dNTP, whereas other analogues have alkaline substitutions at the bridging oxygen. Use of these analogues allows the first systematic comparison of effects of 5'-triphosphate acidity modification on active site structures and the rate constant of DNA synthesis. These ternary complex structures with incoming dATP, dTTP, and dCTP analogues reveal the enzyme's active site is not grossly altered by the acidity modifications of the triphosphate group, yet with analogues of all three incoming dNTP bases, subtle structural differences are apparent in interactions around the nascent base pair and at the guanidinium groups of active site arginine residues. These results are important for understanding how acidity modification of the incoming dNTP's 5'-triphosphate can influence DNA polymerase activity and the significance of interactions at arginines 183 and 149 in the active site.

Figure 1:

A surface image of the DNA polymerase β active site illustrating many of the enzyme-substrate interactions (in green) to be discussed.

Figure 2:. The active site of the DNA polymerase β reference structure (1.8 Å) with a dideoxy primer and incoming dATP.

The nucleotide metal, magnesium (green sphere), is shown, and the catalytic metal site is occupied by sodium (purple sphere). The catalytic and nucleotide metals were assigned as magnesium and sodium, respectively, based on the average coordination distances (shown in Figure S2) and the number of ligands. Coordinating water molecules are shown (small red spheres). Coordination distances (Å) are shown. Key active site amino acid residues are labeled, and the template (Tn) and primer (P) nucleotides are labeled. Two residues distal to the active site are also labeled, R182 and E316.

Figure 3:. Bar diagram of main inter-atomic distances for dATP analogues

The distances that are similar to that of reference structure (dATP) are colored black, distances that are at least 0.2 Å longer, or shorter than that of reference structure are colored red and green respectively. The red horizontal line shows the distance in Å for the structure with the parent compound, with O in the β,γ-bridging position. The descriptions and significance of these measured distances are summarized in Table S2.

Figure 4:. Overlay of active sites of DNA polymerase β with dideoxy primers with incoming dATP (light gray) and difluoro dATP analogue (cyan).

(A) The overlay was made with the 155–260 region of the enzyme. B) Magnification of the overlay in panel A illustrating the contacts around the gamma phosphate group. The CF₂ analogue F atoms are shown in light green.

Figure 5:. Overlay of the active sites of DNA polymerase β with dideoxy primers (p) and incoming dichloro (pink) and difluoro dATP (cyan) analogues.

The CCl₂ analogue Cl atoms are in dark green and the CF₂ analogue F atoms are in light green in the background. E316 no longer makes a contact with R182 in these structures, and R283 does not make a contact with the Tn-1 sugar ring O4 oxygen, as is seen in the reference structure. The nucleotide magnesium atoms are in a different location in these structures, and contacts with Pγ oxygens are different.

Figure 6:. The active site of DNA polymerase β with the incoming A-CHCH₃ analogue of dATP.

The structure is similar to the reference dATP structure, but many contacts are shorter than in the reference structure (see Fig.3), including the primer sugar C3′ and Pα distance (3.7 Å), and in addition, the Pγ oxygens are too far away from R149 to make significant contacts.

Figure 7:. Bar diagram of main inter-atomic distances for dTTP analogues

The distances that are similar to that of reference structure (2FMS) are colored black, distances that are at least 0.2 Å longer, or shorter than that of reference structure are colored red and green respectively. The red horizontal line shows the distance in Å for the reference structure (2FMS). The descriptions and significance of these measured distances are summarized in Table S2.

Figure 8:. Comparisons of F and Cl monohalogen analogue structures of dTTP.

A) Overlay of R-isomer (pink, slow on HPLC), S-isomer (cyan, fast on HPLC), and diastereoisomer mixture (gold) of CHF analogues of dTTP in the active site of DNA polymerase β. B) Overlay of the R-isomer (pink, slow on HPLC), S-isomer (cyan, fast on HPLC), and diastereoisomer mixture (gold) of T-CHCL analogues of dTTP in the active site of pol β. These overlays were made with the 155–260 region of the enzyme. The distances (Å) for the halogen and R183 contact are shown.

Figure 9:. Overlay of active site structures of T-CF₂ (gold) and T-CCL₂ (pink) analogues.

A significant difference in the interaction with R149 is illustrated. A water molecule (red sphere) connects the guanidine group of R149 to the Pγ O3G of the T-CCl₂ analogue structure, but not in the case of the T-CF₂ structure. The two main-chain contacts at G189 are different in the two structures with 3.1 Å in the structure of T-CF₂. The catalytic and nucleotide metals were assigned as magnesium and sodium respectively based on the average coordination distances (shown in Figure S5) and the number of ligands.

Figure 10:. Bar diagram of main inter-atomic distances for dCTP analogues

The distances that are similar to that of reference structure (dCTP) are colored black, distances that are at least 0.2 Å longer, or shorter than that of reference structure are colored red and green respectively. The red horizontal line shows the distance in Å for the reference structure (2FMS). The descriptions and significance of these measured distances are summarized in Table S2.

Figure 11:

Overlay of active sites of DNA polymerase β with incoming dCTP analogues.

Figure 12:. Overlay of active site structures of three CF₂ analogues with bases dA (cyan), dC (gold) and dT (pink).

The different positions of R149 are illustrated along with the distinct intervening water molecule and distances for the guanidine nitrogen and Pγ O3G contact in the C-CF₂ structure. The main-chain contacts with G189 and the contacts with R183 are similar in the three structures. The overlay was made with the 155–260 region of the enzyme.

Scheme 1:

General DNA Polymerase Reaction Pathway