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. 2016 Aug 19;11(8):2158-64.
doi: 10.1021/acschembio.6b00187. Epub 2016 Jun 6.

Conformational States of HIV-1 Reverse Transcriptase for Nucleotide Incorporation vs Pyrophosphorolysis-Binding of Foscarnet

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Conformational States of HIV-1 Reverse Transcriptase for Nucleotide Incorporation vs Pyrophosphorolysis-Binding of Foscarnet

Kalyan Das et al. ACS Chem Biol. .
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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.

Figures

Figure 1
Figure 1. Structural states of HIV-1 RT trapped in crystals of RT/apt-DNA
The crystals of an RT/apt-DNA complex (I) were soaked with AZTTP and CaCl2 for the formation of an RT/apt-DNA/AZTTP complex (II); N and P sites are indicated in I. Crystals of I were soaked with AZTTP and MgCl2 for catalytic addition of AZTMP resulting in complex III. Crystals of III were further soaked with PFA and MgCl2 for the formation of the RT/apt-DNA/PFA complex (IV).
Fig. 2
Fig. 2. Binding and incorporation of AZT-TP by RT in crystals
A. AZTTP (yellow) is bound at the polymerase active site in RT/apt-DNA/AZTTP complex (II). B. The ternary structures of RT/apt-DNA/AZTTP and RT/DNA/AZTTP (PDB ID. 3V4I; cyan) are highly superimposable at the polymerase active site. C. The polymerase active site region after incorporation of AZTMP in the RT/apt-DNA (AZTMP terminated) N-site structure (III). D. Location of incorporated AZTMP with respect to the catalytic triad of aspartates.
Figure 3
Figure 3. Binding of PFA
A. A schematic view of transition at the polymerase active site upon binding of PFA (from complex III to IV). B. Difference Fo - Fc (magenta) and 2Fo - Fc (blue) electron density maps at 3 Å resolution revealed the PFA binding mode and polymerase active-site conformation. C. Superposition of structures II and IV shows the positional differences of chelating oxygen atoms of PFA compared to the pyrophosphate moiety (β and γ phosphates) of AZTTP. Comparison of the binding modes of PFA with HIV-1 RT (D) and with engineered chimeric DNA polymerase UL54D (E). F. Interactions of PFA with RT. The sites of PFA-resistance mutations K65R and A114S are in cyan. G. Electrostatic potential surface of RT and location of PFA in the complex.
Figure 3
Figure 3. Binding of PFA
A. A schematic view of transition at the polymerase active site upon binding of PFA (from complex III to IV). B. Difference Fo - Fc (magenta) and 2Fo - Fc (blue) electron density maps at 3 Å resolution revealed the PFA binding mode and polymerase active-site conformation. C. Superposition of structures II and IV shows the positional differences of chelating oxygen atoms of PFA compared to the pyrophosphate moiety (β and γ phosphates) of AZTTP. Comparison of the binding modes of PFA with HIV-1 RT (D) and with engineered chimeric DNA polymerase UL54D (E). F. Interactions of PFA with RT. The sites of PFA-resistance mutations K65R and A114S are in cyan. G. Electrostatic potential surface of RT and location of PFA in the complex.
Figure 4
Figure 4. Binding of α-T-CNP and PFA to RT
A. Superposition of α-T-CNP (cyan) in the RT/DNA cross-linked complex (PDB Id. 4R5P) on to the RT/apt-DNA/α-T-CNP complex (inhibitor, yellow; protein, gray). The current structure reveals coordination of α-T-CNP with both Mg2+ ions. B. Active site superposition of structures IV and V shows the relative positioning and metal chelation of PFA (green) and α-T-CNP (yellow); only the chelating oxygen atoms are colored red.

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