Conformational flexibility in the flap domains of ligand-free HIV protease

Abstract

The crystal structures of wild-type HIV protease (HIV PR) in the absence of substrate or inhibitor in two related crystal forms at 1.4 and 2.15 A resolution are reported. In one crystal form HIV PR adopts an 'open' conformation with a 7.7 A separation between the tips of the flaps in the homodimer. In the other crystal form the tips of the flaps are 'curled' towards the 80s loop, forming contacts across the local twofold axis. The 2.3 A resolution crystal structure of a sixfold mutant of HIV PR in the absence of substrate or inhibitor is also reported. The mutant HIV PR, which evolved in response to treatment with the potent inhibitor TL-3, contains six point mutations relative to the wild-type enzyme (L24I, M46I, F53L, L63P, V77I, V82A). In this structure the flaps also adopt a 'curled' conformation, but are separated and not in contact. Comparison of the apo structures to those with TL-3 bound demonstrates the extent of conformational change induced by inhibitor binding, which includes reorganization of the packing between twofold-related flaps. Further comparison with six other apo HIV PR structures reveals that the 'open' and 'curled' conformations define two distinct families in HIV PR. These conformational states include hinge motion of residues at either end of the flaps, opening and closing the entire beta-loop, and translational motion of the flap normal to the dimer twofold axis and relative to the 80s loop. The alternate conformations also entail changes in the beta-turn at the tip of the flap. These observations provide insight into the plasticity of the flap domains, the nature of their motions and their critical role in binding substrates and inhibitors.

Amide hydrogen exchange in HIV–1 subtype B and C proteases – insights into reduced drug susceptibility and dimer stability

“…Salt bridges also affect the conformational stability of a macromolecule [35,36]. Crystal structure analysis shows that the C-SA protease (PDB ID: 3U71) [37] lacks the K20-E34 and E35-R57 salt bridges compared with the subtype B protease (PDB ID: 2PC0) [13] in their crystalline forms. Therefore, the reduced number of ionic interactions and a less stable terminal b-sheet evident in the C-SA protease are major determinants for the apparent overall reduced conformational stability of the C-SA protease.…”

“…This mutation is associated with virological failure or a reduced virological response to atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, saquinavir and tipranavir in combination with ritonavir [42][43][44][45][46][47][48][49]. Mutations at residue position 89 are associated with virological failure to darunavir and tipranavir in combination with ritonavir [44,50], and mutations at resi- In the subtype B protease (A) (PDB ID: 2PC0 [13]), M36 appears to provide anchorage for E35, and stabilizes the salt bridge between E35 and R57. I36 in the C-SA protease (B) (PDB ID: 3U71 [37]) is implicated in fewer hinge region interactions and disruption of the E35-R57 salt bridge.…”

“…2A). Structural and protease dynamics studies indicate that the curled conformer may be an intermediate state that allows formation of the fully open conformer [9,11,13]. Entry of a substrate or inhibitor to the active site of the protease requires substantial movement of the flaps (approximately 15 A from their position in the closed conformer) [14].…”

“…1. Polymorphic sites of the subtype B protease (PDB ID: 2PC0) [13] and the C-SA protease (PDB ID: 3U71) [37]. The flexible flaps of the protease (residues at positions [46][47][48][49][50][51][52][53][54] are coloured cyan.…”

Generation of Infectious Feline Immunodeficiency Virus (FIV) Encoding FIV/Human Immunodeficiency Virus Chimeric Protease

“…Comparisons of the 3 dimensional structures of the two PRs led to the rational design of TL-3, a broad-based PR inhibitor capable of blocking infection by FIV, simian immunodeficiency virus (SIV), and HIV-1, as well as many drug-resistant HIV-1 variants (10,12,21,23,31,32). Similar approaches comparing the structures of FIV and drug-resistant HIV-1 PRs to that of WT HIV-1 PR have led to the development of additional PR-inhibiting compounds with broadened efficacy (8,9,19,29,41,42).…”