The crystal structure of HIV-1 RT bound to double stranded DNA (PDB code 2hmi). HIV-1 RT functions as heterodimer of p66 and p51 subunits. Due to the resemblance of p66 to a closed right hand, subdomains of p66 have been named as the ‘palm’ (red), fingers (blue), and thumb (green). The p66 subdomain contains two active sites, the polymerase and the RNase H active sites (orange). The region between the RNase H and polymerase active sites is known as the connection (yellow) subdomain. The p51 (dark brown) subunit is derived from the proteolytic cleavage of RNase H from p66 and has identical primary and secondary structure. However, the tertiary structure of p51 is markedly different than p66 leading to a non-functional arrangement of catalytic residues. The template/primer (white/gray) is seen in the DNA-binding cleft formed primarily by the p66 subunit of the enzyme. Figures 1, 2, 3, 7, 9 and 11 were generated using PyMOL [4].

Interactions of RNA/DNA and DNA/DNA template/primers with HIV-1 RT. The two different template/primers (cyan/red) bind in the nucleic acid binding cleft of RT in a similar way. The RNA/DNA (panel A; PDB code 1hys) maintains the protein contacts seen in the complex with DNA/DNA (panel B, PDB code 2hmi) (these contacts are not shown in panel A. and has thirteen additional contacts. Nine of these contacts are through the 2′-OH group of the RNA sugar backbone (blue), whereas four are mediated through phosphate-backbone of RNA/DNA (plum). In panel B, the template-protein contacts are colored yellow and the primer-protein contacts are colored magenta.

A. The effect of sugar ring conformation on catalysis of DNA synthesis by HIV-1 RT. Sugar-ring conformation of the nucleotide at the 3′-primer end (panel A). For efficient DNA synthesis to occur, the sugar ring conformation of the nucleotide at the 3′-primer end should be in the north (2′-exo/3′-endo) conformation (panel A, shown in yellow). The south (2′-endo/3′-exo) conformation (panel A, shown in cyan) of the sugar ring at the primer terminus mispositions the primer 3′-OH away for an in-line nucleophilic attack on the α-phosphate of the incoming dNTP (green), thereby resulting in inefficient catalysis. B. The sugar-ring conformation of the incoming dNTP should be north (panel B, green). If the incoming dNTP or nucleotide analog were to have a south conformation (panel B, cyan) this would result in steric hindrance with the aromatic ring of Y115 (shown in red). Thus, the favored conformation of the incoming dNTP is the north conformation (panel B, green). The software Coot [55] was used to prepare various sugar ring conformations of the primer terminus and incoming dNTP, starting from the structural coordinates of the HIV-1 RT/DNA/dNTP ternary complex (PDB Code 1rtd).

Stereo-view of the polymerase active site of HIV-1 RT. The primer strand is shown in gray, the incoming dNTP in orange, and the RT active-site residues interacting with substrates or metal ions in cyan. Metal ions A and B are shown as magenta bullets. The Cα-traces of the p66 palm and fingers subdomains are shown in red and blue, respectively. The yellow dotted lines depict the coordination geometry of the metal ions and the interactions of p66 fingers-subdomain residues with the incoming dNTP. The coordination geometry of metal ion B (also known as structural metal ion) is octahedral. Due to lack of the primer 3′OH group in the crystal structure (PDB file 1rtd), the coordination of metal ion A (known as catalytic metal) is incomplete. Interactions of fingers-subdomain residues K65, R72, and Q151 with dNTP are also shown in yellow dotted lines. Figures 4, 5, and 7 were generated by MolMol [58].

Conformational changes of p66 fingers and thumb subdomains during DNA synthesis by HIV-1 RT. Similar to other nucleic acid polymerases, HIV-1 RT undergoes conformational changes at various steps of the catalytic cycle. In the unliganded HIV-1 RT (E, shown as red tracing), the fingers and thumb subdomains fold over the active site to render it inaccessible. Binding of the template/primer opens up the fingers and thumb subdomains to accommodate the DNA/DNA or RNA/DNA substrates (green tracing). The binding of incoming dNTP causes the p66 fingers subdomain to move to a closed form and trap dNTP in a catalytically competent conformation (cyan tracing). After incorporation of dNMP, release of PPi, and translocation of the elongated template/primer, HIV-1 RT assumes the conformation seen in enzyme-DNA bound structure (green).

Nucleoside inhibitors (NRTIs) of HIV-1 RT: Chemical structures of NRTIs. With the exception of EFdA, all NRTIs lack a 3′ OH moiety. After their incorporation into DNA by HIV-1 RT, they act as chain terminators. In contrast, EFdA contains a 3′-OH and acts as a translcoation-defective reverse transcriptase inhibitor. FTC is 5-fluoro derivative of 3TC; the latter has not been included in this figure. Chemical structures were drawn with Chem Sketch 3.5 [82].

Stereo-view of the NNRTI binding site. NNRTIs bind in a pocket formed primarily by hydrophobic residues, which are rendered as cyan sticks with N and O atoms colored blue and red, respectively. A recently approved NNRTI, etravirine, is shown in gray. The p66 palm, thumb, and connection subdomains are shown in red, green, and yellow, respectively. Structural coordinates are from PDB code 1sv5.

Mechanism of NNRTI inhibition: NNRTI-binding alters the geometry of the p66 thumb subdomain, changes the position of the ‘primer grip’, and causes misalignment of important components at the polymerase active site. The structure of RT in complex with TIBO (cyan and gray ribbons) is superposed onto the DNA-bound RT (red) to demonstrate the shift in the position of ‘primer grip’ as a result of NNRTI binding. Residues Y181 and D185 are shown as reference points for the NNRTI binding pocket and YMDD loop, respectively. The TIBO inhibitor is shown as orange balls and sticks, whereas the template/primer (gray/blue) is shown as ribbons and plates.

Structural components of NRTI resistance by the excision mechanism: Molecular model showing the binding of ATP (red) to AZT-resistant HIV-1 RT and unblocking of AZTMP-terminated primer. Van der Waals surfaces are drawn for polymerase active site residues (magenta) and residues involved in ATP binding and AZT resistance (yellow). Mutated amino acids M41L, K70R, L210W, and T215Y are shown with black labels. The two terminal nucleotide base pairs of the template/primer are shown. The 3′ end of the primer is AZTMP and is positioned at the N site. AZT-resistant enzyme has the amino acid substitutions M41L, D67N, K70R, T215Y, and K219Q. Y215 is likely to have aromatic interactions with the purine ring of ATP (red). R70 is likely to interact with the ribose ring and/or the α-phosphate of ATP. The wild-type amino acids at K219 and D67 were retained in the figure to show a potential salt bridge between the residues.

The NNRTI binding pocket (NNIBP) in various RT/NNRTI complexes. Clockwise from top left: RT/Etravirine (PDB Code 1sv5), RT/nevirapine (1vrt), RT/delavirdine (1klm), and RT/efavirenz (1ikw). Mutations conferring resistance to these drugs occur primarily in and around the NNIBP. Residue D185 of the YMDD loop is labeled as a polymerase active site reference point. The p66 palm, thumb, and connection subdomains are shown in red, green, and yellow, respectively.

Binding of TMC-278 at the NNIBP results in a conformational change of residues Y181 and Y188. In panel A, comparison of unliganded RT (green ribbon and side chains) and RT/TMC-278 complex (blue ribbon and white side chains) shows that the aromatic rings of Y181 and Y188 “flip” as a result of TMC-278 binding to HIV-1 RT. Panel B shows the comparison of WT (blue ribbon, white side chains, and gray TMC-278) and Y181C/K103N (cyan ribbon, yellow side chains, and orange TMC-278) RT complexes with TMC-278. The yellow side chains belong to the TMC-278 bound K103N mutant RT. This figure also shows that the loss of interaction due to Y181C mutation is compensated by the interaction between cyanovinyl group and conserved Y183 (based on structures from pdb codes: 1dlo, 2zd1 and 3bgr).