Folded Monomer of HIV-1 Protease

Abstract

The mature human immunodeficiency virus type 1 protease rapidly folds into an enzymatically active stable dimer, exhibiting an intricate interplay between structure formation and dimerization. We now show by NMR and sedimentation equilibrium studies that a mutant protease containing the R87K substitution (PR R87K ) within the highly conserved Gly 86 -Arg 87 -Asn 88 sequence forms a monomer with a fold similar to a single subunit of the dimer. However, binding of the inhibitor DMP323 to PR R87K produces a stable dimer complex. Based on the crystal structure and our NMR results, we postulate that loss of specific interactions involving the side chain of Arg 87 destabilizes PR R87K by perturbing the inner C-terminal ␤-sheet (residues 96 -99 from each monomer), a region that is sandwiched between the two ␤-strands formed by the N-terminal residues (residues 1-4) in the mature protease. We systematically examined the folding, dimerization, and catalytic activities of mutant proteases comprising deletions of either one of the terminal regions (residues 1-4 or 96 -99) or both. Although both N-and C-terminal ␤-strands were found to contribute to dimer stability, our results indicate that the inner Cterminal strands are absolutely essential for dimer formation. Knowledge of the monomer fold and regions critical for dimerization may aid in the rational design of novel inhibitors of the protease to overcome the problem of drug resistance. Human immunodeficiency virus type 1 (HIV-1)1 is a 99-residue aspartic acid protease and is active as a homodimer. The interface of the free protease dimer is stabilized through interactions between the two subunits at the active site and at the termini (1, 2). The protease catalyzes its own release from the Gag-Pol polyprotein in addition to the maturation of the virally encoded structural proteins and replication enzymes required for the assembly and production of viable virions (3). Thus, the protease has served as one of the primary targets for the development of drugs against AIDS. Structure-based design of drugs targeted against the wild-type mature protease has aided in the development of several potent inhibitors that are specific for binding to the active site (4). Although several of these drugs are in clinical use and have curtailed the progression of the disease, the effectiveness of long-term treatment has been restricted due to naturally selected protease variants exhibiting lower affinity for the drugs than the wild-type enzyme. Various drug-resistant mutants of the protease have been identified (5). Different resistance mechanisms based on the observed structural changes in drug-resistant mutants have been proposed. In general, the mutants modulate structure and interactions within the active site as well as inter-and intrasubunit flexibility (6 -8).Because of the difficulty in designing active-site inhibitors that avoid the problem of drug resistance, the target region for drug development has been extended to areas in which no drug-selected mutations have occurred to date...

Revisiting Monomeric HIV-1 Protease

“…The 1 H-15 N HSQC spectrum of PR T26A is similar to those of PR-(1-95) and PR R87K monomers (Fig. 3B) (16), demonstrating that PR T26A is also a folded monomer. 15 N NMR relaxation measurements on PR R87K in the absence of inhibitor DMP323 revealed significant motions on the subnanosecond time scale for the N-and C-terminal residues 2-10 and 93-99 unique to the monomer (16).…”

“…Disrupting the D29/R87 Interaction Destabilizes Dimer Formation-We previously demonstrated that a protease construct bearing the conservative mutation R87K does not exhibit dimer formation, even at concentrations up to ϳ1 mM (16). This suggested that an interaction involving the side chain of Arg 87 , most likely the one between Arg 87 and Asp 29 , stabilized the dimer, possibly by anchoring the ␣-helix and thereby correctly positioning the two C-terminal strands of the ␤-sheet.…”

“…The proteins were folded according to the procedure previously described (16). The concentrations of purified proteins were determined spectrophotometrically.…”

“…2). Studies on PR-(1-95), PR-(5-99), and PR-(5-95) demonstrated that even though both the N-and C-terminal strands contributed to dimer stability, interactions between the C-terminal residues 96 -99 that form the inner strands of the sheet were absolutely essential for dimer formation at protease concentrations below 1 mM (16).…”

“…These residues are located in the helical stretch of the structure, connecting the main body of the protein to the last ␤-strand of the terminal ␤-sheet (6). This motif appears to constitute a crucial structural element, because even a conservative mutation, R87K, disrupted dimerization of the mature protease (15,16). Interestingly, substrates with micromolar affinity to protease did not restore the dimeric form of PR R87K , as evidenced by its poor catalytic activity.…”