Caught in the Act: the 1.5 A resolution crystal structures of the HIV-1 protease and the I54V mutant reveal a tetrahedral reaction intermediate

Abstract

HIV-1 protease (PR) is the target for several important antiviral drugs used in AIDS therapy. The drugs bind inside the active site cavity of PR where normally the viral polyprotein substrate is bound and hydrolyzed. We report two high-resolution crystal structures of wild-type PR (PRWT) and the multi-drug-resistant variant with the I54V mutation (PRI54V) in complex with a peptide at 1.46 and 1.50 A resolution, respectively. The peptide forms a gem-diol tetrahedral reaction intermediate (TI) in the crystal structures. Distinctive interactions are observed for the TI binding in the active site cavity of PRWT and PRI54V. The mutant PRI54V/TI complex has lost water-mediated hydrogen bond interactions with the amides of Ile50 and Ile50' in the flap. Hence, the structures provide insight into the mechanism of drug resistance arising from this mutation. The structures also illustrate an intermediate state in the hydrolysis reaction. One of the gem-diol hydroxide groups in the PRWT complex forms a very short (2.3 A) hydrogen bond with the outer carboxylate oxygen of Asp25. Quantum chemical calculations based on this TI structure are consistent with protonation of the inner carboxylate oxygen of Asp25', in contrast to several theoretical studies. These TI complexes and quantum calculations are discussed in relation to the chemical mechanism of the peptide bond hydrolysis catalyzed by PR.

Figure 1

HIV-1 PR homodimer structure in cartoon representation showing the secondary structure. The tetrahedral intermediate (gray), Asp25, Asp25′ (yellow), and site of mutation Ile54 to Val (cyan) are shown in ball-and-stick mode.

Figure 2

The stereo view of the omit electron density (F_o-F_c) for the tetrahedral intermediate in the PR_WT/TI structure. Contour levels are 2.2 σ for (a) and 3.6 σ for (b). The higher contour level in (b) shows the positions of the TI hydroxyl oxygens.

Figure 3

Hydrogen bond interactions of the tetrahedral intermediate with wild-type PR (a) and PR_I54V mutant (b) residues. Hydrogen bonds are indicated by dotted lines.

Figure 3

Hydrogen bond interactions of the tetrahedral intermediate with wild-type PR (a) and PR_I54V mutant (b) residues. Hydrogen bonds are indicated by dotted lines.

Figure 4

Superposition of PR_WT/TI (colored magenta) and PR_I54V/TI (colored by atom type) structures. The largest shifts occur for residues 50′ and 81 as indicated by arrows. The C-H…O contact of 3.5 Å between the main-chain carbonyl of Ile50′ and the Cγ2 of Ile54 is maintained in the mutant structure, while the hydrophobic contacts of the Ile50′ side-chain with Pro80 and the polar interaction of the Ile50′ amide with the water are elongated.

Figure 5

Hydrogen bonds in the active site of (a) PR_WT/TI complex, (b) PR_I54V/TI complex, and (c) unliganded PR_F53L dimer. Only part of the TI molecule that includes residues Thr and Leu flanking the gem-diol moiety is shown in panels a and b for clarity.

Figure 6

Superposition of active sites in PR_F53L (colored magenta), PR_WT/TI (colored by atom type) and PR_WT/DRV (colored cyan) structures. The shifts of Asp25, Asp25′ and TI, DRV relative to the unliganded structure are indicated by black arrows. The figure was created by aligning main-chain atoms for the corresponding protein structures.

Figure 7

Positions of hydrogen atoms in the active site of HIV-1 PR according to DFT calculations. The models with hydrogen on the inner carboxylate oxygen are stabilized by 12 and 28 kcal/mol), respectively, for the ligand-free (a) and TI-complex (c) relative to the alternate models (b and d).

Figure 8

Proposed reaction mechanism of the peptide bond hydrolysis by HIV-1 PR.