Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase.
Bottom Line: Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years.These structures reveal that relative to D-dCTP, each of these L-nucleotides has its sugar ring rotated by 180° with an unusual O4'-endo sugar puckering and exhibits multiple triphosphate-binding conformations within the active site of the polymerase.Such rare binding modes significantly decrease the incorporation rates and efficiencies of these L-nucleotides catalyzed by the polymerase.
Affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.Show MeSH
Related in: MedlinePlus
Mentions: Notably, the distance between the α-phosphorus atom of an incoming nucleotide and the primer 3′-OH is significantly shorter for Type-B conformation of (–)FTC-PPNP (3.3 Å) than in Type-A conformation of (–)FTC-PPNP (5.7 Å) and (–)3TC-PPNP (5.6 Å) (Figure 2). Among Type-A and Type-B conformations, the latter is more similar to the chair-like conformation based on the orientation of the α-phosphate group (Figure 4). In addition, the interactions between residues Arg51 and Lys159 with the γ-phosphate group of (–)FTC-PPNP only existed in its Type-A conformation (Figure 5A), not in Type-B (Figure 5B). This suggests that transition of the triphosphate moiety of (–)FTC-PPNP to canonical chair-like conformation (Figure 4A) from the goat tail-like one (Figure 5B) was easier than from the N-shaped conformation (Figure 5A). Taken together, we propose the following productive order for the triphosphate's binding conformation: chair-like >> Type-B > Type-A. Based on the similar kp values of (–)3TC-TP and (–)FTC-TP (Table 1), (–)3TC-TP also likely adopted Type-B conformation during catalysis although this minor species was not captured during crystallization. In addition, Type-B conformation of (–)FTC-PPNP was stabilized by the presence of a weak hydrogen bond (3.2 Å) between its α-phosphate and the F5 atom of its nitrogenous base (Figure 2D). Such an interaction should not exist in the presumed Type-B conformation of (–)3TC-PPNP. The presence or absence of this intramolecular interaction altered the abundance of Type-B conformation of the L-analogs in solution and likely contributed to their differential incorporation efficiency with Dpo4 (Table 1) and HIV-1 RT (52).
Affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.