Effect of flap mutations on structure of HIV-1 protease and inhibition by saquinavir and darunavir

Abstract

HIV-1 (human immunodeficiency virus type 1) protease (PR) and its mutants are important antiviral drug targets. The PR flap region is critical for binding substrates or inhibitors and catalytic activity. Hence, mutations of flap residues frequently contribute to reduced susceptibility to PR inhibitors in drug-resistant HIV. Structural and kinetic analyses were used to investigate the role of flap residues Gly48, Ile50, and Ile54 in the development of drug resistance. The crystal structures of flap mutants PR(I50V) (PR with I50V mutation), PR(I54V) (PR with I54V mutation), and PR(I54M) (PR with I54M mutation) complexed with saquinavir (SQV) as well as PR(G48V) (PR with G48V mutation), PR(I54V), and PR(I54M) complexed with darunavir (DRV) were determined at resolutions of 1.05-1.40 A. The PR mutants showed changes in flap conformation, interactions with adjacent residues, inhibitor binding, and the conformation of the 80s loop relative to the wild-type PR. The PR contacts with DRV were closer in PR(G48V)-DRV than in the wild-type PR-DRV, whereas they were longer in PR(I54M)-DRV. The relative inhibition of PR(I54V) and that of PR(I54M) were similar for SQV and DRV. PR(G48V) was about twofold less susceptible to SQV than to DRV, whereas the opposite was observed for PR(I50V). The observed inhibition was in agreement with the association of G48V and I50V with clinical resistance to SQV and DRV, respectively. This analysis of structural and kinetic effects of the mutants will assist in the development of more effective inhibitors for drug-resistant HIV.

Figure 1

(a) The chemical structures of saquinavir and darunavir. (b) Structure of HIV-1 PR dimer with the locations of mutated residues Gly48 (cyan), Ile50 (red), Ile54 (green) indicated by spheres for main chain atoms in both subunits. Darunavir is shown in sticks colored by atom type. The flap residues (45–55) and the 80’s loop (78–82) are colored in blue and purple, respectively.

Figure 2

The Fo-Fc omit maps showing the mutated residues and the flap residues (47–54) contoured at 3.3 sigma. Val 48 is from PR_G48V-DRV, Val50 from PR_I50V-SQV, Val54 from PR_I54V-SQV, Met54 from PR_I54M-SQV and flap residues from PR_I54M-SQV. The cyan sticks indicate the alternate conformations of main-chain and side-chain atoms in Val50 and Met54.

Figure 3

The Fo-Fc omit maps of saquinavir (upper panel) and darunavir (lower panel) contoured at 3.3 sigma. Saquinavir is colored by atom type from complex PR_I54V-SQV. Darunavir is from complex PR_I54V-DRV showing alternate conformations of 60/40% occupancy. The major conformation is colored by atom type and the minor is colored pink.

Figure 4

The flap regions for superimposed complexes with darunavir. The wild type PR is in green, PR_G48V in cyan, PR_I54V in magenta and PR_I54M in red. The arrows indicate the shifts between Cα atoms of PR_G48V and of the wild type PR with distances in Å.

Figure 5

PR_I54M interactions with inhibitor. (a) The major orientation of saquinavir. (b) The major orientation of darunavir. Hydrogen bonds are indicated in red, CH-π interactions in blue and C-H…O in purple. Interatomic distances are shown in Å. Note that the water-mediated interaction of the NH2 of darunavir with the Asp30 side chain is replaced by a direct hydrogen bond in the wild type PR and the other mutant structures.

Figure 6

(a) Comparison of the flaps of PR_G48V-DRV and of wild type PR-DRV in the two subunits. (b) Selected PR_G48V interactions with darunavir in the minor conformation. The mutant structures are colored by atom type and wild type PR is in grey. Dashed lines indicate van der Waals interactions with interatomic distances shown in Å. The arrows show the shift between the two structures with distances in Å.

Figure 6

(a) Comparison of the flaps of PR_G48V-DRV and of wild type PR-DRV in the two subunits. (b) Selected PR_G48V interactions with darunavir in the minor conformation. The mutant structures are colored by atom type and wild type PR is in grey. Dashed lines indicate van der Waals interactions with interatomic distances shown in Å. The arrows show the shift between the two structures with distances in Å.

Figure 7

The interactions of residues 50, 51, 54 and 79–81 in (a) PR_I50V-SQV, (b) PR_I54M-DRV, and (c) PR_I54V-DRV. The structures of mutants are colored by atom types and the corresponding structures of wild type PR are grey. Dashed lines indicate van der Waals interactions with interatomic distances shown in Å. The arrows show the shift between the two structures with distances in Å.

Figure 7

The interactions of residues 50, 51, 54 and 79–81 in (a) PR_I50V-SQV, (b) PR_I54M-DRV, and (c) PR_I54V-DRV. The structures of mutants are colored by atom types and the corresponding structures of wild type PR are grey. Dashed lines indicate van der Waals interactions with interatomic distances shown in Å. The arrows show the shift between the two structures with distances in Å.