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. (Fig. 1), possess L-stereochemistry. Both lamivudine and emtricitabine have been shown to be more effective in inhibiting HIV-1 RT and less toxic than their enantiomeric D-isomers (1-4). In addition, both lamivudine, a potent inhibitor of hepatitis B virus (HBV) (5), and telbivudine, the L-analog of thymidine, are approved drugs for the treatment of HBV infection, whereas emtricitabine is currently in clinical trials for this purpose (6). These L-nucleoside analogs demonstrate less clinical toxicity than their corresponding D-isomers, likely because human DNA polymerases possess strong D-stereoselectivity by preferentially binding and incorporating D-dNTPs over unnatural nucleotides with L-stereochemistry (L-dNTPs) during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by any DNA polymerase or RT has not been established, although D-stereoselectivity has been successfully explored in antiviral drug development.Despite high clinical efficacy, NRTIs are often associated with various drug toxicities resulting from the inhibition of host DNA polymerases that share a catalytic mechanism akin to HIV-1 RT (7). There are 16 identified human DNA polymerases that belong to A-, B-, X-, and Y-families. NRTI-associated mitoc...

Structural insights into the recognition of nucleoside reverse transcriptase inhibitors by HIV‐1 reverse transcriptase: First crystal structures with reverse transcriptase and the active triphosphate forms of lamivudine and emtricitabine

“…In fact, (−)FTC‐TP and (−)3TC‐TP are similarly effective against RT compared to their chiral pair (+)FTC‐TP/(+)3TC‐TP, but they are incorporated much less efficiently into Pol γ . Similar stereochemical preferences between D‐ and L‐ stereoisomers of nucleic acids were also reported for Polβ and Polλ . To date, there are no structural data available for (−)/(+)FTC‐TP and (−)3TC‐TP in complex with RT.…”

“…As the different enantiomer in complex with Pol γ did not result in substantial conformational changes in the binding mode of the compounds, it was predicted that the binding mode of (−)FTC‐TP and (−)3TC‐TP in RT also should have been similar to the one of (+)FTC‐TP/TTP . An additional insight into underlying reasons for such shifted binding modes of (−)FTC‐TP and (−)3TC‐TP in RT can be obtained by considering structural comparisons with the DNA damage repair enzymes, Pol λ and Pol β . In those cases, multiple (−)FTC‐TP and (−)3TC‐TP binding modes are observed for these DNA Pols in contrast to the natural dCTP substrate.…”

“…NRTIs target the active site of the reverse transcriptase (RT) protein, a critical enzyme for the replication cycle of HIV, and they function as chain terminators of the viral DNA replication upon their incorporation in the DNA primer . However, several studies have linked the long‐term usage of the NRTIs to mitochondrial dysfunction and, additionally, several other studies have reported the correlation between mitochondrial toxicity, genomic instability, and the off‐target effect of NRTIs toward several human DNA polymerases (Pols) such as Pol γ . The design of highly selective NRTIs is challenging as human DNA Pols and RT share a similar mechanism of nucleotide incorporation and discrimination .…”

“…The design of highly selective NRTIs is challenging as human DNA Pols and RT share a similar mechanism of nucleotide incorporation and discrimination . Previous studies have shown that some Pols are more susceptible to NRTIs toxicity than others and have suggested that particular attention needs to be paid in developing and understanding of how natural nucleoside and NRTIs in their 5′‐triphosphate form discriminate between the host Pols, especially Pol γ …”

“…(+)FTC-TP is the enantiomer of (−)FTC-TP additionally, several other studies have reported the correlation between mitochondrial toxicity, genomic instability, and the off-target effect of NRTIs toward several human DNA polymerases (Pols) such as Pol γ. [4][5][6][7] The design of highly selective NRTIs is challenging as human DNA Pols and RT share a similar mechanism of nucleotide incorporation and discrimination. 8 Previous studies have shown that some Pols are more susceptible to NRTIs toxicity than others and have suggested that particular attention needs to be paid in developing and understanding of how natural nucleoside and NRTIs in their 5 0 -triphosphate form discriminate between the host Pols, especially Pol γ.…”