5I3U

STRUCTURE OF HIV-1 REVERSE TRANSCRIPTASE N-SITE COMPLEX; CATALYTIC INCORPORATION OF AZTMP to A DNA aptamer in CRYSTAL

Summary for 5I3U

• Entry DOI: 10.2210/pdb5i3u/pdb
• Related: 5D3G 5HP1 5HRO 5I42
• Related PRD ID: PRD_900003
• Descriptor: HIV-1 REVERSE TRANSCRIPTASE P66 SUBUNIT, HIV-1 REVERSE TRANSCRIPTASE P51 SUBUNIT, DNA (39-MER), ... (6 entities in total)
• Functional Keywords: rt, dna aptamer, foscavir, n site complex, pyrophosphate, pyrophosphorolysis, phosphonoformic acid, pfa, 2-o-methylcytidine, p51, p66, transferase, transferase-dna complex, transferase/dna
• Biological source: Human immunodeficiency virus type 1 group M subtype B (isolate BH10) (HIV-1); More
• Total number of polymer chains: 6
• Total formula weight: 256680.35

Authors

Das, K.,Arnold, E. (deposition date: 2016-02-11, release date: 2016-06-01, Last modification date: 2023-09-27)

Primary citation

Das, K.,Balzarini, J.,Miller, M.T.,Maguire, A.R.,DeStefano, J.J.,Arnold, E.
Conformational States of HIV-1 Reverse Transcriptase for Nucleotide Incorporation vs Pyrophosphorolysis-Binding of Foscarnet.
Acs Chem.Biol., 11:2158-2164, 2016

PubMed Abstract: HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3'-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg(2+) ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg(2+) (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors.

PubMed: 27192549
DOI: 10.1021/acschembio.6b00187

Experimental method

X-RAY DIFFRACTION (3 Å)