Revealing the dimer dissociation and existence of a folded monomer of the mature HIV-2 protease.
Purification and in vitro protein-folding schemes were developed to produce monodisperse samples of the mature wild-type HIV-2 protease (PR2), enabling a comprehensive set of biochemical and biophysical studies to assess the dissociation of the dimeric protease. An E37K substitution in PR2 significantly retards autoproteolytic cleavage during expression. Furthermore, it permits convenient measurement of the dimer dissociation of PR2(E37K) (elevated K(d) approximately 20 nM) by enzyme kinetics. Differential scanning calorimetry reveals a T(m) of 60.5 for PR2 as compared with 65.7 degrees C for HIV-1 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the T(m) of PR2 increases by 14.8 degrees C in the presence of DRV as compared with 22.4 degrees C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2(T26A)) or a deletion of the C-terminal residues 96-99 (PR2(1-95)), drastically increase the K(d) (>10(5)-fold). PR2(T26A) and PR2(1-95) consist predominantly of folded monomers, as determined by nuclear magnetic resonance (NMR) and size-exclusion chromatography coupled with multiangle light scattering and refractive index measurements (SMR), whereas wild-type PR2 and its active-site mutant PR2(D25N) are folded dimers. Addition of twofold excess active-site inhibitor promotes dimerization of PR2(T26A) but not of PR2(1-95), indicating that subunit interactions involving the C-terminal residues are crucial for dimer formation. Use of SMR and NMR with PR2 facilitates probing for potential inhibitors that restrict protein folding and/or dimerization and, thus, may provide insights for the future design of inhibitors to circumvent drug resistance.
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, Maryland 20892-0520, USA. johnl@intra.niddk.nih.gov
John M
JM
Louis
Rieko
R
Ishima
Annie
A
Aniana
Jane M
JM
Sayer
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D000069454:Darunavir; D016333:HIV Protease / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000201:enzymology; D015498:HIV-2 / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D055503:Protein Multimerization; D016415:Sequence Alignment; D013449:Sulfonamides / Q000494:pharmacology
D017320:HIV Protease Inhibitors; D013449:Sulfonamides; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1; C065184:p16 protease, Human immunodeficiency virus 2; D000069454:Darunavir