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Revealing the dimer dissociation and existence of a folded monomer of the mature HIV‐2 protease
Author(s) -
Louis John M.,
Ishima Rieko,
Aniana Annie,
Sayer Jane M.
Publication year - 2009
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.261
Subject(s) - dimer , chemistry , darunavir , protease , hiv 1 protease , active site , crystallography , enzyme kinetics , stereochemistry , enzyme , biochemistry , human immunodeficiency virus (hiv) , biology , organic chemistry , antiretroviral therapy , viral load , immunology
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 ∼20 n M ) by enzyme kinetics. Differential scanning calorimetry reveals a T m of 60.5 for PR2 as compared with 65.7°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°C in the presence of DRV as compared with 22.4°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.