title: Structural basis for the binding and incorporation of nucleotide analogs with L-stereochemistry by human DNA polymerase λ.
abstract: Although lamivudine and emtricitabine, two L-deoxycytidine analogs, have been widely used as antiviral drugs for years, a structural basis for D-stereoselectivity against L-dNTPs, enantiomers of natural nucleotides (D-dNTPs), by any DNA polymerase or reverse transcriptase has not been established due to lack of a ternary structure of a polymerase, DNA, and an incoming L-dNTP. Here, we report 2.10-2.25 Å ternary crystal structures of human DNA polymerase λ, DNA, and L-deoxycytidine 5'-triphosphate (L-dCTP), or the triphosphates of lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) with four ternary complexes per asymmetric unit. The structures of these 12 ternary complexes reveal that relative to D-deoxycytidine 5'-triphosphate (D-dCTP) in the canonical ternary structure of Polλ-DNA-D-dCTP, L-dCTP, (-)3TC-TP, and (-)FTC-TP all have their ribose rotated by 180°. Among the four ternary complexes with a specific L-nucleotide, two are similar and show that the L-nucleotide forms three Watson-Crick hydrogen bonds with the templating nucleotide dG and adopts a chair-like triphosphate conformation. In the remaining two similar ternary complexes, the L-nucleotide surprisingly interacts with the side chain of a conserved active site residue R517 through one or two hydrogen bonds, whereas the templating dG is anchored by a hydrogen bond with the side chain of a semiconserved residue Y505. Furthermore, the triphosphate of the L-nucleotide adopts an unprecedented N-shaped conformation. Our mutagenic and kinetic studies further demonstrate that the side chain of R517 is critical for the formation of the abovementioned four complexes along proposed catalytic pathways for L-nucleotide incorporation and provide the structural basis for the D-stereoselectivity of a DNA polymerase.
authors.affiliation: Department of Chemistry and Biochemistry and.
authors.forename: Rajan
authors.initials: R
authors.lastname: Vyas
authors.affiliation: Department of Chemistry and Biochemistry andThe Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Zahurancik
authors.affiliation: Department of Chemistry and Biochemistry andThe Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210 suo.3@osu.edu.
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D019943:Amino Acid Substitution; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D020125:Mutation, Missense; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D003845:Deoxycytosine Nucleotides; C415367:DNA polymerase beta2; D019951:DNA Polymerase beta
keywords: DNA polymerase lambda; DNA polymerase stereoselectivity; L-nucleotide:arginine paring; antiviral nucleoside analogs; pre–steady-state kinetics