Mechanism of dissociative inhibition of HIV protease and its autoprocessing from a precursor.
Dimerization is indispensible for release of the human immunodeficiency virus protease (PR) from its precursor (Gag-Pol) and ensuing mature-like catalytic activity that is crucial for virus maturation. We show that a single-chain Fv fragment (scFv) of a previously reported monoclonal antibody (mAb1696), which recognizes the N-terminus of PR, dissociates a dimeric mature D25N PR mutant with an enhanced dimer dissociation constant (K(d)) in the sub-micromolar range to form predominantly a monomer-scFv complex at a 1:1 ratio, along with small (5-10%) amounts of a dimer-scFv complex. Enzyme kinetics indicate a mixed mechanism of inhibition of the wild-type PR, which exhibits a K(d)<10nM, with effects both on K(m) and k(cat) at an scFv-to-PR ratio of 10:1. ScFv binds to the N-terminal peptide P(1)QITLW(6) of PR and to PR monomers with dissociation constants of ≤30 nM and ~100 nM, respectively. Consistent with an ~400-fold increase in the dissociation of the antibody (K(Ab)) on even addition of an acetyl group to P(1) of the peptide, the antibody fails to inhibit N-terminal autoprocessing of the PR from a model precursor (at ~5 μM). However, subsequent to this cleavage, it sequesters the PR, thus blocking autoprocessing at its C-terminus. A second monoclonal antibody [PRM1 (human monoclonal antibody to PR)], which recognizes part of the flap region (residues 41-47) of the mature PR and its precursor, does not inhibit autoprocessing and ensuing catalytic activity. However, its failure to recognize drug-resistant clinical mutants of PR may be beneficial to monitor the selection of mutations in this region under drug pressure.
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA.
Jane M
JM
Sayer
Annie
A
Aniana
John M
JM
Louis
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D019281:Dimerization; D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D057127:Single-Chain Antibodies; D016333:HIV Protease