title: Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease.
abstract: Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR-DMP323 complex, and distinctively for PRD25N-DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer-inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: johnl@niddk.nih.gov.
authors.forename: John M
authors.initials: JM
authors.lastname: Louis
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA. Electronic address: julien.roche@nih.gov.
authors.forename: Julien
authors.initials: J
authors.lastname: Roche
mesh_terms: D005075:Biological Evolution; D020134:Catalytic Domain / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D016333:HIV Protease / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1
keywords: HIV protease; calorimetry; drug resistance; high-pressure NMR; protein folding landscape