Kinetic, stability, and structural changes in high-resolution crystal structures of HIV-1 protease with drug-resistant mutations L24I, I50V, and G73S

Abstract

The crystal structures, dimer stabilities, and kinetics have been analyzed for wild-type human immunodeficiency virus type 1 (HIV-1) protease (PR) and resistant mutants PR(L24I), PR(I50V), and PR(G73S) to gain insight into the molecular basis of drug resistance. The mutations lie in different structural regions. Mutation I50V alters a residue in the flexible flap that interacts with the inhibitor, L24I alters a residue adjacent to the catalytic Asp25, and G73S lies at the protein surface far from the inhibitor-binding site. PR(L24I) and PR(I50V), showed a 4% and 18% lower k(cat)/K(m), respectively, relative to PR. The relative k(cat)/K(m) of PR(G73S) varied from 14% to 400% when assayed using different substrates. Inhibition constants (K(i)) of the antiviral drug indinavir for the reaction catalyzed by the mutant enzymes were about threefold and 50-fold higher for PR(L24I) and PR(I50V), respectively, relative to PR and PR(G73S). The dimer dissociation constant (K(d)) was estimated to be approximately 20 nM for both PR(L24I) and PR(I50V), and below 5 nM for PR(G73S) and PR. Crystal structures of the mutants PR(L24I), PR(I50V) and PR(G73S) were determined in complexes with indinavir, or the p2/NC substrate analog at resolutions of 1.10-1.50 Angstrom. Each mutant revealed distinct structural changes relative to PR. The mutated residues in PR(L24I) and PR(I50V) had reduced intersubunit contacts, consistent with the increased K(d) for dimer dissociation. Relative to PR, PR(I50V) had fewer interactions of Val50 with inhibitors, in agreement with the dramatically increased K(i). The distal mutation G73S introduced new hydrogen bond interactions that can transmit changes to the substrate-binding site and alter catalytic activity. Therefore, the structural alterations observed for drug-resistant mutations were in agreement with kinetic and stability changes.

Figure 1

PR dimer structure (green ribbons) with indinavir (red bonds). The sites of mutation are indicated by black spheres for Leu24, Ile50 and Gly73. Only one subunit is labeled.

Figure 2

Protease stability. (a) Sensitivity to urea: L24I, filled circles and continuous line (UC₅₀ = 1.05M); I50V, open squares and dotted line (UC₅₀ = 0.97 M); G73S, open circles and continuous line (UC₅₀ = 1.54M). (b) Dimer dissociation: L24I, filled circles and continuous line (K_d = 22 nM); I50V, open squares and dotted line (K_d = 19 nM); G73S, open circles (no dissociation observed).

Figure 3

Omit map for indinavir in the crystal structure of PR_L24I–IDV contoured at a level of 3.5σ.

Figure 4

(a) Residues with alternate conformations. Alternate conformations of residues in six dimers of five new crystal structures. Alternate conformations for both side-chain and main-chain atoms were included. (b) Omit maps for mutated residues contoured at a level of 3.5σ. Val50′ in PR_I50V–IDV had a single conformation for the side-chain. Two alternate conformations are shown for the side-chains of Val 50 (relative occupancy of 0.7/0.3) in PR_I50V–IDV, Ile24 (relative occupancy of 0.6/0.4) in PR_L24I–p2/NC, and Ser73 (relative occupancy of 0.5/0.5) in PR_G73S–IDV structures.

Figure 5

Structural differences at sites of mutation. Broken lines indicate hydrogen bond interactions (2.6–3.3 Å). Dotted lines indicate van der Waals interactions (3.5–4.2 Å). Dash-dot lines indicate distances over 4.2 Å for comparison. (a) Interactions of Leu/Ile24 with Leu90 and Phe99′ in the indinavir complexes. PR is green and PR_L24I is red. (b) Interactions of Ile/Leu 50 with Ile 47′ and Ile 84′ in the complexes with p2/NC. PR is in green and PR_I50V is in red. (c) Interactions of Gly73 with residues 74, 31, 29 and 88 in PR-IDV structure. (d) Interactions of Ser73 with residues 74, 31, 29 and 88 in the second dimer of PR_G73S-IDV crystal structure. The side-chain of Ser73′ has two conformations.

Figure 6

Protease-inhibitor interactions. Only the residues involved in hydrogen bond interactions are shown. Water molecules are represented as spheres. Hydrogen bonds are indicated by broken lines. (a) PR_L24I hydrogen bond interactions with indinavir. Water molecules are labeled A–D. (b) Interactions of Arg8′ with the pyridyl group of indinavir in PR_I50V–IDV. The omit map is contoured at 3.5σ. (c) PR_L24I interactions with p2/NC. Water molecules are labeled 1–8. Arg8 and Arg8′ are omitted for clarity. (d) Selected interactions of the side-chains of Ile/Leu50 and 50′ with indinavir in the PR_I50V and PR indinavir complexes. PR-IDV is in green and PR_I50V–IDV is red. Only the central portion of indinavir is shown with van der Waals contacts indicated by dotted lines with distances in Å.

Figure 7

The catalytic site of PR_L24I–p2/NC at 1.1 Å resolution. The 2F_o – F_c map is in green and contoured at 2.6σ, and the positive F_o – F_c map is in purple, contoured at 3.5σ.