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Comparative Study
. 2010 Sep;277(18):3699-714.
doi: 10.1111/j.1742-4658.2010.07771.x. Epub 2010 Aug 2.

Amprenavir complexes with HIV-1 protease and its drug-resistant mutants altering hydrophobic clusters

Chen-Hsiang Shen  1 , Yuan-Fang Wang, Andrey Y Kovalevsky, Robert W Harrison, Irene T Weber
Affiliations
Comparative Study

Amprenavir complexes with HIV-1 protease and its drug-resistant mutants altering hydrophobic clusters

Chen-Hsiang Shen et al. FEBS J. 2010 Sep.

Abstract

The structural and kinetic effects of amprenavir (APV), a clinical HIV protease (PR) inhibitor, were analyzed with wild-type enzyme and mutants with single substitutions of V32I, I50V, I54V, I54M, I84V and L90M that are common in drug resistance. Crystal structures of the APV complexes at resolutions of 1.02-1.85 Å reveal the structural changes due to the mutations. Substitution of the larger side chains in PR(V32I) , PR(I54M) and PR(L90M) resulted in the formation of new hydrophobic contacts with flap residues, residues 79 and 80, and Asp25, respectively. Mutation to smaller side chains eliminated hydrophobic interactions in the PR(I50V) and PR(I54V) structures. The PR(I84V)-APV complex had lost hydrophobic contacts with APV, the PR(V32I)-APV complex showed increased hydrophobic contacts within the hydrophobic cluster and the PR(I50V) complex had weaker polar and hydrophobic interactions with APV. The observed structural changes in PR(I84V)-APV, PR(V32I)-APV and PR(I50V)-APV were related to their reduced inhibition by APV of six-, 10- and 30-fold, respectively, relative to wild-type PR. The APV complexes were compared with the corresponding saquinavir complexes. The PR dimers had distinct rearrangements of the flaps and 80's loops that adapt to the different P1' groups of the inhibitors, while maintaining contacts within the hydrophobic cluster. These small changes in the loops and weak internal interactions produce the different patterns of resistant mutations for the two drugs.

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Figures

Figure 1
Figure 1
(a) The chemical structures of amprenavir (APV) and saquinavir (SQV). (b) Structure of HIV-1 PR dimer with the sites of mutation Val32, Ile50, Ile54, Ile84 and Leu90 indicated by green sticks for side chain atoms in both subunits. Amino acids are labeled in one subunit only. APV is shown in magenta sticks. The amino acids in the inner hydrophobic cluster are indicated by numbered red spheres, and the amino acids in the outer hydrophobic cluster are shown as blue spheres.
Figure 1
Figure 1
(a) The chemical structures of amprenavir (APV) and saquinavir (SQV). (b) Structure of HIV-1 PR dimer with the sites of mutation Val32, Ile50, Ile54, Ile84 and Leu90 indicated by green sticks for side chain atoms in both subunits. Amino acids are labeled in one subunit only. APV is shown in magenta sticks. The amino acids in the inner hydrophobic cluster are indicated by numbered red spheres, and the amino acids in the outer hydrophobic cluster are shown as blue spheres.
Figure 2
Figure 2
Inhibitor Binding Site in PRWT-APV. a) APV and PR residues in the binding site with alternate conformations. Omit maps for major (green) and minor (magenta) conformations of APV, interacting PR residues Asp25, Gly48 and Asp30 from both subunits, and the conserved flap water are contoured at a level of 3.5 σ. b) Hydrogen bond, C-H···O and H2O···π interactions between PR (gray) and APV (cyan). Hydrogen bond interactions are indicated by dashed lines. C-H···O and H2O···π interactions are indicated by dotted lines.
Figure 2
Figure 2
Inhibitor Binding Site in PRWT-APV. a) APV and PR residues in the binding site with alternate conformations. Omit maps for major (green) and minor (magenta) conformations of APV, interacting PR residues Asp25, Gly48 and Asp30 from both subunits, and the conserved flap water are contoured at a level of 3.5 σ. b) Hydrogen bond, C-H···O and H2O···π interactions between PR (gray) and APV (cyan). Hydrogen bond interactions are indicated by dashed lines. C-H···O and H2O···π interactions are indicated by dotted lines.
Figure 3
Figure 3
The interactions of mutated residues in (a) PRV32I-APV, (b) PRI50V-APV, (c) PRI54M-APV, (d) PRI54V-APV, (e) PRI84V-APV, and (f) PRl90M-APV. The grey color corresponds to wild type PR-APV and the cyan color indicates the mutant complex. Dashed lines indicate van der Waals interactions and dotted lines show C-H···O interactions. Interatomic distances are shown in Å with black lines indicating the PRWT and red lines indicating the mutant. Interatomic distances of > 4.3 Å are shown in dash-dot lines to indicate the absence of favorable interaction.
Figure 4
Figure 4
Structural differences between APV and SQV complexes. (a) The root mean square (RMS) difference (Å) per residue is plotted for Cα atoms of SQV complexes compared with the corresponding APV complexes: PRWT (blue line), PRI50V (red line) and PRI54V (green line). (b) Comparison of the flap regions in the structures. The complexes with APV are in cyan, and the complexes with SQV are in grey. The arrow indicates the shifts between Cα atoms at the residues 50 and 51 in the PR complexes with the two inhibitors. (c) The width across the S1-S1′ subsites increases in PRWT-SQV relative to PRWT-APV. Similar changes were seen for the mutant complexes, except for PRI50V.
Figure 5
Figure 5
Interactions of Ile50, Ile54′ and Thr80′. (a) PRWT-APV compared to PRWT-SQV. (b) PRI50V-APV compared to PRI50V-SQV. (c) PRI54M-APV compared to PRI54M-SQV. (d) PRI54V-APV compared to PRI54V-SQV. (e) PRI84V-APV compared to PRI84V-SQV. Dashed lines indicate van der Waals contacts with interatomic distances in Å. Dotted lines indicate C-H···O interactions. Black lines indicate interactions in SQV complexes. Red lines indicate interactions in APV complexes.
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