PDBsum entry 1c9r

Transferase/immune system/DNA

PDB id: 1c9r

Contents

Protein chains

558 a.a.

430 a.a.

214 a.a.

220 a.a.

DNA/RNA

References listed in PDB file

Key reference

Title

Lamivudine (3tc) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-Branched amino acids.

Authors

S.G.Sarafianos, K.Das, A.D.Clark, J.Ding, P.L.Boyer, S.H.Hughes, E.Arnold.

Ref.

Proc Natl Acad Sci U S A, 1999, 96, 10027-10032. [DOI no: 10.1073/pnas.96.18.10027]

PubMed id

10468556

Abstract

An important component of triple-drug anti-AIDS therapy is 2', 3'-dideoxy-3'-thiacytidine (3TC, lamivudine). Single mutations at residue 184 of the reverse transcriptase (RT) in HIV cause high-level resistance to 3TC and contribute to the failure of anti-AIDS combination therapy. We have determined crystal structures of the 3TC-resistant mutant HIV-1 RT (M184I) in both the presence and absence of a DNA/DNA template-primer. In the absence of a DNA substrate, the wild-type and mutant structures are very similar. However, comparison of crystal structures of M184I mutant and wild-type HIV-1 RT with and without DNA reveals repositioning of the template-primer in the M184I/DNA binary complex and other smaller changes in residues in the dNTP-binding site. On the basis of these structural results, we developed a model that explains the ability of the 3TC-resistant mutant M184I to incorporate dNTPs but not the nucleotide analog 3TCTP. In this model, steric hindrance is expected for NRTIs with beta- or L- ring configurations, as with the enantiomer of 3TC that is used in therapy. Steric conflict between the oxathiolane ring of 3TCTP and the side chain of beta-branched amino acids (Val, Ile, Thr) at position 184 perturbs inhibitor binding, leading to a reduction in incorporation of the analog. The model can also explain the 3TC resistance of analogous hepatitis B polymerase mutants. Repositioning of the template-primer as observed in the binary complex (M184I/DNA) may also occur in the catalytic ternary complex (M184I/DNA/3TCTP) and contribute to 3TC resistance by interfering with the formation of a catalytically competent closed complex.

Figure 2.
Fig. 2. Superposition of the polymerase active sites of wild-type HIV-1 RT/DNA/Fab and M184I HIV-1 RT/DNA/Fab. The wild-type RT complex is shown in white, the mutant RT complex in cyan. The wild-type and M184I RT structures were superimposed on the basis of the core of the p66 palm subdomains (residues 107 to 112 and 151 to 215 of their corresponding p66 subunits).

Figure 3.
Fig. 3. Ribbon diagram of the superimposed polymerase active sites of wild-type HIV-1 RT/DNA/Fab and M184I HIV-1 RT/DNA/Fab. The wild-type protein and DNA are shown in gray, the mutant protein in red, and DNA in the mutant RT/DNA complex in yellow.

Secondary reference #1

Title

Structure and functional implications of the polymerase active site region in a complex of HIV-1 rt with a double-Stranded DNA template-Primer and an antibody FAB fragment at 2.8 a resolution.

Authors

J.Ding, K.Das, Y.Hsiou, S.G.Sarafianos, A.D.Clark, A.Jacobo-Molina, C.Tantillo, S.H.Hughes, E.Arnold.

Ref.

J Mol Biol, 1998, 284, 1095-1111. [DOI no: 10.1006/jmbi.1998.2208]

PubMed id

9837729

Figure 1.
Figure 1. Ribbon [Carson 1987] diagram showing the overall structure of the HIV-1 RT/dsDNA/Fab28 complex. The subdomains of the p66 and p51 subunits of HIV-1 RT are colored as follows: fingers, blue; palm, red; thumb, green; connection, yellow; and RNase H, orange. The bound dsDNA is shown with the template strand as a dark gray ribbon and the primer strand as a light gray ribbon; base-pairs are represented by bars. The monoclonal antibody fragment Fab28 is shown with the light chain in light gray and the heavy chain in dark gray.

Figure 3.
Figure 3. (a) Structure of the polymerase active site region of HIV-1 RT including the primer grip. Secondary structural elements of the p66 palm subdomain are shown as red ribbons. The three catalytically essential aspartic acid residues (Asp110, Asp185, and Asp186) are shown with cyan side-chains. Tyr183 and Met184, which form part of the conserved YMDD motif, are shown with gold side-chains. Amino acid residues at the primer grip are shown in green. The dsDNA is shown with the template strand in dark gray and the primer strand in light gray. (b) A schematic diagram showing interactions between the 3′-terminal nucleotide of the primer strand (Pri1) and amino acid residues at the polymerase active site, with selected distances given in Å. Hydrogen-bonding interactions between the side-chain O^δ1 atom of Asp185 and the 3′-OH of Pri1, and between the amide nitrogen atom of Met230 of the primer grip and the phosphate oxygen atom of Pri1 are indicated by heavy lines.

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #2

Title

Touching the heart of HIV-1 drug resistance: the fingers close down on the dntp at the polymerase active site.

Authors

S.G.Sarafianos, K.Das, J.Ding, P.L.Boyer, S.H.Hughes, E.Arnold.

Ref.

Chem Biol, 1999, 6, R137.

PubMed id

10322129