HIV-1 Protease with 20 Mutations Exhibits Extreme Resistance to Clinical Inhibitors through Coordinated Structural Rearrangements

Abstract

The escape mutant of HIV-1 protease (PR) containing 20 mutations (PR20) undergoes efficient polyprotein processing even in the presence of clinical protease inhibitors (PIs). PR20 shows >3 orders of magnitude decreased affinity for PIs darunavir (DRV) and saquinavir (SQV) relative to PR. Crystal structures of PR20 crystallized with yttrium, substrate analogue p2-NC, DRV, and SQV reveal three distinct conformations of the flexible flaps and diminished interactions with inhibitors through the combination of multiple mutations. PR20 with yttrium at the active site exhibits widely separated flaps lacking the usual intersubunit contacts seen in other inhibitor-free dimers. Mutations of residues 35–37 in the hinge loop eliminate interactions and perturb the flap conformation. Crystals of PR20/p2-NC contain one uninhibited dimer with one very open flap and one closed flap and a second inhibitor-bound dimer in the closed form showing six fewer hydrogen bonds with the substrate analogue relative to wild-type PR. PR20 complexes with PIs exhibit expanded S2/S2′ pockets and fewer PI interactions arising from coordinated effects of mutations throughout the structure, in agreement with the strikingly reduced affinity. In particular, insertion of the large aromatic side chains of L10F and L33F alters intersubunit interactions and widens the PI binding site through a network of hydrophobic contacts. The two very open conformations of PR20 as well as the expanded binding site of the inhibitor-bound closed form suggest possible approaches for modifying inhibitors to target extreme drug-resistant HIV.

The Role of Select Subtype Polymorphisms on HIV-1 Protease Conformational Sampling and Dynamics

“…9B). A similar absence of the salt bridge pattern and turned away hinge loop pattern is seen in the recently reported x-ray structure of a clinically isolated multidrug-resistant construct PR20, which contains E35D (49). It is likely that the Glu-35/Arg-57 salt bridge may hold this loop into a more structured conformation in subtype B, where loss of this interaction may result in an increase in backbone dynamics and lead to a more flexible mode of flap motion, resulting in the novel fourth curled open or asymmetric flap conformation.…”

“…Additionally, these substitutions are commonly seen as secondary mutations, which are believed to restore enzyme catalytic function after drug-resistant primary mutations develop (48,49). As shown in Fig.…”

“…Bridge Formation Patterns-To provide molecular level structural evidence to support our hypothesis that the population in DEER distance profiles ϳ27 Å is a novel conformer rarely seen in molecular dynamics simulations of subtype B, and to explore how this mutation may structurally influence HIV-1 PR, we scrutinized several x-ray crystal structures, including subtype B (51,66,68), C (69,70), subtype F (66), CRF01_A/E (8), and other mutants with (49,71) and without the E35D mutation. We found, as shown in Fig.…”

A Cluster Analysis on the Structural Diversity of Protein Crystals, Exemplified by Human Immunodeficiency Virus Type 1 Protease

“…It should be noted that all of the crystal structures in group 5 were provided by two research groups. [29][30][31] The L63P mutation that is an amino substitution sometimes appeared in drug-resistant viruses 32,33) is seen in every cluster except for group 3.…”

“…9) Human immunodeficiency virus type 1 (HIV-1) protease is one of the most extensively studied viral proteins because of its importance as a target of antiviral drugs. 4,5) Since inhibition of the enzymatic activity hinders maturation of the viral precursor and leads to incomplete replication of the virus, inhibitors of HIV-1 protease are effective in chemotherapy for HIV-1 infectious diseases.…”