SNAP annotations: PDB entry 8vbi

PDB-id

8vbi; DSSR-derived features in text and JSON formats; DNAproDB

Class

transcription, transferase-DNA

Method

cryo-EM (2.3 Å)

Summary

Kinetic intermediate states of hiv-1 rt DNA synthesis captured by cryo-EM

Reference

Vergara S, Zhou X, Santiago U, Alaoui-El-Azher M, Conway JF, Sluis-Cremer N, Calero G (2024): "Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription." Nat Commun, 15, 10553. doi: 10.1038/s41467-024-54618-y.

Abstract

Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg₂₊ (site B) inducing formation of a negatively charged pocket where a second floating Mg₂₊ can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg₂₊ and the primer 3'-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication.