Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6 angstroms resolution crystal structures of HIV-1 protease mutants with substrate analogs

Abstract

HIV-1 protease (PR) and two drug-resistant variants--PR with the V82A mutation (PR(V82A)) and PR with the I84V mutation (PR(I84V))--were studied using reduced peptide analogs of five natural cleavage sites (CA-p2, p2-NC, p6pol-PR, p1-p6 and NC-p1) to understand the structural and kinetic changes. The common drug-resistant mutations V82A and I84V alter residues forming the substrate-binding site. Eight crystal structures were refined at resolutions of 1.10-1.60 A. Differences in the PR-analog interactions depended on the peptide sequence and were consistent with the relative inhibition. Analog p6(pol)-PR formed more hydrogen bonds of P2 Asn with PR and fewer van der Waals contacts at P1' Pro compared with those formed by CA-p2 or p2-NC in PR complexes. The P3 Gly in p1-p6 provided fewer van der Waals contacts and hydrogen bonds at P2-P3 and more water-mediated interactions. PR(I84V) showed reduced van der Waals interactions with inhibitor compared with PR, which was consistent with kinetic data. The structures suggest that the binding affinity for mutants is modulated by the conformational flexibility of the substrate analogs. The complexes of PR(V82A) showed smaller shifts of the main chain atoms of Ala82 relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. PR(V82A) was able to compensate for the loss of interaction with inhibitor caused by mutation, in agreement with kinetic data, but substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation. Hence, these structures help to explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.

Fig. 1

Electron density map of HIV-1 protease with the V82A mutation (PR_V82A)–p2-NC crystal structure. The 2Fo–Fc map was contoured at a level of 2.2σ. Hydrogen bond interactions are shown with distances in Å. (A) Residues 78–82. (B) Asp30 interacting with P2′ Gln.

Fig. 2

Residues with alternate conformations. The number of occurrences of alternate conformations for each residue in the A and B subunits of the eight crystal structures are shown.

Fig. 3

Superposition of the wild type HIV-1 protease (PR), PR with the V82A mutation (PR_V82A) and PR_V82A in complex with p2-NC. The ribbons represent the backbones of the dimers and the p2-NC analog. The sites of mutations Val82 and Ile84 are shown by red bonds for PR, blue for Ala82, and green for Val84 in both subunits.

Fig. 4

Superposition of four complexes of HIV-1 protease with the V82A mutation (PR_V82A) with the inhibitors CA-p2, p2-NC, p6^pol-PR and p1-p6.

Fig. 5

Hydrogen bond interactions between protein and inhibitor. Hydrogen bond interactions are shown for interatomic distances of 2.5– 3.3 Å. Water molecules are indicated by red spheres. Water-mediated hydrogen bonds are shown as red dashed lines, while direct interactions between the protease and inhibitor are in black. (A) Hydrogen bond interactions between HIV-1 protease with the V82A mutation (PR_V82A) and CA-p2. (One water-mediated interaction between P3 Arg and Pro 81′ is not shown.) (B) Hydrogen bond interactions between PR and p2-NC. (Water-mediated interactions of both termini of inhibitor with Arg8 and 8′ are not shown.) (C) Hydrogen bond interactions between PR and p6^pol-PR. (Water-mediated interactions of the C termini of p6^pol-PR with Asp60 and Gln61 are not shown.) (D) Hydrogen bond interactions between PR_V82A and p1-p6. (Water-mediated interactions of the C termini of p1-p6 with Trp6, Arg8 and Arg87′ are not shown.)

Fig. 6

Structural variation around residues 8184 in p2-NC, p6^pol-PR, p1-p6, UIC-94017 and indinavir complexes. The protease (PR) structure is shown in purple, PR with the 184V mutation (PR_I84V) in green and PR with the V82A mutation (PR_V82A) in blue bonds. Interatomic distances (Å) are indicated as dashed lines. (A) PR_V82A–p2-NC superimposed on PR–p2-NC. (B) PR_I84V–p2-NC superimposed on PR–p2-NC. (C) PR_V82A–p6^pol-PR superimposed on PR–p6^pol-PR. (D) PR_V82A–p1-p6 superimposed on PR–p1-p6. (E) PR_V82A–UIC-94017 superimposed on PR–UIC-94017. (F) PR_V82A–indinavir superimposed on PR–indinavir.

Fig. 7

Structural variation around the active site. (A) PR–p1-p6 is shown (colored by atom type) superimposed on D25N–p1-p6 (1KJF) in green bonds. Distances within 4.0 Å are shown. (B) PR– UIC-94017 is shown as yellow bonds superimposed on PR–p1-p6 complex (colored by atom type).

Fig. 8

Electron density maps at the active site of the PR_V82A– p2-NC complex. The 2Fo-Fc map is green and was contoured at a level of 2.2, whereas the Fo-Fc map is contoured at 3.2 and colored purple for positive.