Unique Flap Conformation in an HIV-1 Protease with High-Level Darunavir Resistance

Abstract

Darunavir (DRV) is one of the most powerful protease inhibitors (PIs) for treating human immunodeficiency virus type-1 (HIV-1) infection and presents a high genetic barrier to the generation of resistant viruses. However, DRV-resistant HIV-1 infrequently emerges from viruses exhibiting resistance to other protease inhibitors. To address this resistance, researchers have gathered genetic information on DRV resistance. In contrast, few structural insights into the mechanism underlying DRV resistance are available. To elucidate this mechanism, we determined the crystal structure of the ligand-free state of a protease with high-level DRV resistance and six DRV resistance-associated mutations (including I47V and I50V), which we generated by in vitro selection. This crystal structure showed a unique curling conformation at the flap regions that was not found in the previously reported ligand-free protease structures. Molecular dynamics simulations indicated that the curled flap conformation altered the flap dynamics. These results suggest that the preference for a unique flap conformation influences DRV binding. These results provide new structural insights into elucidating the molecular mechanism of DRV resistance and aid to develop PIs effective against DRV-resistant viruses.

Keywords: Darunavir; HIV-1 protease; I50V; crystal structure; drug resistance; flap; molecular dynamics simulation; protease inhibitor.

Figure 1

The PI susceptibilities of 20 clinical HIV-1 isolates. The arrows in this figure highlight the results for FS5929. The “fold resistance” in IC₅₀ are given relative to the IC₅₀ values for the reference wild-type HIV-1 JR-CSF, according to the equation, fold resistance = (the IC₅₀ against a clinical isolate)/(the IC₅₀ against the JR-CSF).

Figure 2

In vitro selection to induce DRV-resistant viruses. The selection of FS5929 and HIV-1 JR-CSF are indicated with gray dotted and black solid lines, respectively. The arrow indicates the sampling time point for the collection of FS5929R, which we focused on in this study. The x-axis and y-axis represent “days after the initial viral infection to cell culture” and “DRV concentration (μM) in cell culture medium,” respectively.

Figure 3

Crystal structure of FS5929R1 PR. (A) Ribbon diagram of the PR, which forms a homodimer. The α-helices and β-strands are colored in red and yellow, respectively. The flap regions (residues 43–58) are highlighted in orange. (B) Locations of mutated residues on the PR. The red, yellow, and green spheres indicate the locations of the major DRV resistance, minor DRV resistance, and other mutations, respectively, that appeared after the DRV-resistance induction experiment using FS5929. Mutations that originally existed in FS5929 are shown in gray spheres. (C) Highlighted structure around the flaps in the PR. The mutations are shown as sticks. The mutations V32I, I47V, and I50V, which appeared during the in vitro selection, are highlighted with red letters. (D) Superposition of our crystal structures of the PRs (gray) with the crystal structures of the open form [PDB IDs: 2PC0 (cyan), 1TW7 (purple), 4NPU (orange), and 3UF3 (navy)]. (E) Superposition of our crystal structures of the PRs with a crystal structure of the semi-open form [PDB IDs: 1HHP (green)].

Figure 4

Comparison of the crystal structures of ligand-free PRs; closed-form (PDB ID: 1HVR), semi-open form (1HHP), open form (2PC0, 1TW7, 4NPU, and 3UF3), and the PR purified in this study. (A) RMSD calculations comparing the crystal structures by using the coordinates of the Cα atoms. (B) Comparison of each Cα atom in the PR structures with the corresponding Cα atom in the FS5929R1 PR.

Figure 5

Flap dynamics in the crystal structure and MD simulations. (A) B factors of the respective residues in the FS5929R1 PR estimated from the crystal structure and MD simulations. (B) The minimum distance between two flaps in the WT and FS5929R1 PRs calculated with an in-house program. (C) The time course of the distance between the V50 Cα and the T80 Cα (left, red) or between the V50′ Cα and the T80′ Cα (left, green) and their histogram (center). The black and gray solid lines indicate the distances in the crystal structure from our study and an open-form structure (PDB ID: 3UF3), respectively. The structural positions of the V50 and T80 Cαs (red) in one monomer and the V50′ and T80′ Cαs in a second monomer are highlighted on the right side of the figure of the PR structure.

Figure 6

Representative structures of simulations of each PR with DRV. (A) Interactions between PR and DRV. The orange dotted lines indicate hydrogen bonds. (B) Superposition onto the WT PR structure. Stick representations show important residues in WT (gray), FS5929R1 (purple), and FS5929 PR (blue) structures. The red arrow indicates the flap region shifted outward in the FS5929R1 PR with DRV.

Figure 7

Hydrogen bond network between PR and DRV predicted from MD simulations. The residues in PR and DRV are highlighted with blue and black, respectively. Hydrogen bonds are represented by dotted lines colored according to their occurrence during the 5.0–6.0 ns simulations, as shown in the bottom bar.