Effect of the Active Site D25N Mutation on the Structure, Stability, and Ligand Binding of the Mature HIV-1 Protease

Abstract

All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR D25N ) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 ؎ 0.09 M) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR D25N in the relatively mobile P1 loop (residues 79 -83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3°C, respectively. Only minimal differences are observed in high resolution crystal structures of PR D25N complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH 2 (RPB), as compared with PR⅐DRV and PR⅐RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T m is smaller for PR D25N (6.2°C) than for PR (8.7°C). The T m of PR D25N ⅐DRV increases by only 3°C relative to free PR D25N , as compared with a 22°C increase for PR⅐DRV, and the mutation increases the ligand dissociation constant of PR D25N ⅐DRV by a factor of ϳ10 6 relative to PR⅐DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.In HIV-1, 2 the protease is synthesized as part of a 165-kDa polyprotein (Gag-Pol). Gag-Pol comprises the matrix, capsid, P2, nucleocapsid, transframe, protease (PR), reverse transcriptase, and integrase domains (1). The protease mediates its own release and the processing of the viral polyproteins, Gag and Gag-Pol, into the necessary structural and functional proteins (1-3). This spatio-temporally regulated process is crucial for the maturation and propagation of HIV (4 -7). Because of this vital role, the mature protease dimer has proven to be a successful target for the development of antiviral agents. Structure-based design of drugs targeted against the mature protease has aided in the development of potent inhibitors that bind specifically to the active site (8, 9). Although several of these inhibitors are in clinical use and have curtailed the progression of the disease, the effectiveness of long term treatment has been limited due to naturally selected protease variants exhibiting lower affinity to the drugs than the wild-type enzyme, and this has been a challenge for the past decade (10). In recent years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens, which include the highly active retroviral therapy, to overcome the problem of drug resistance and curb progress of the disease (11, 12).The HIV-1 protease is composed of 99 amino acids and is a member of the family of aspartic ac...

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

“…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.…”

“…2A, positions 35, 37, and 41 lie within the elbow/hinge region. For EPR and NMR investigations, we and others (50,51), often inactivate HIV-1PR through the D25N active site mutation, which limits self-proteolysis. The DSC measurements serve to demonstrate how protein stability and protein complex stability are altered by the PR5 mutations and D25N-inactivating mutation.…”

“…For HIV-1 PR, the coupled unfolding and dimer stability of the protein or protease-inhibitor complex is reported by the transition midpoint T m (51,52). Fig.…”

Modulation of Human Immunodeficiency Virus Type 1 Protease Autoprocessing by Charge Properties of Surface Residue 69

“…To evaluate further the charge effect of H69 on protease maturation, we altered the parental H to K, L, Q, and E, individually. GST-TFR-PR D25N -FLAG, in which the Asp residue critical for catalytic activity was mutated to Asn (7,11,17), was also constructed to produce the full-length GST fusion precursor serving as a negative control. Mature protease and precursors were detected with anti-FLAG antibody by Western blotting (Fig.…”

In-Silico Assessment of Various PDB Entries of Pfldh Enzyme for their Use in SBDD

“…The 3D structures of chain A's of 1T2D, 1T24, 1U4O, 1U5A, 1XIV, 2A94 and 4B7U ( Figure 1) were then analysed for structure assessment by ANOLEA [7][8][9][10][11][12][13].…”