ABSTRACT
The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation. We have produced monoclonal antibodies specific for the HIV-1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme's activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterized two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognize epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms of protease inhibition by these antibodies.
Subject(s)
Antibodies, Monoclonal/pharmacology , HIV Protease Inhibitors/immunology , HIV Protease/immunology , Animals , Antibodies, Monoclonal/chemistry , HIV Antibodies/chemistry , HIV Antibodies/pharmacology , HIV Protease/chemistry , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/pharmacology , HIV-1/enzymology , HIV-1/immunology , In Vitro Techniques , Mice , Models, Molecular , Molecular Structure , Protein ConformationABSTRACT
BACKGROUND: Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimerization to the active enzymatic form. RESULTS: We report here the crystal structure at 2.7 A resolution of a recombinant single-chain Fv fragment of mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. CONCLUSION: A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the design of inhibitors aimed at binding inactive monomeric species.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/immunology , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/pharmacology , HIV Protease/chemistry , HIV Protease/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/metabolism , Aspartic Acid Endopeptidases/metabolism , Binding Sites, Antibody , Cross Reactions , Crystallography, X-Ray , HIV Protease/metabolism , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Models, Chemical , Models, Molecular , Peptide Fragments/immunology , Peptide Fragments/metabolism , Protein ConformationABSTRACT
We describe a 5-year-old boy with a unique congenital cataract caused by deposition of numerous birefringent, pleiochroic and macroscopically prismatic crystals. Crystal analysis with subsequent automatic Edman degradation and matrix-associated laser desorption ionization time-of-flight mass spectrometry have identified the crystal-forming protein as gammaD-crystallin (CRYGD) lacking the N-terminal methionine. Sequencing of the CRYGD gene has shown a heterozygous C-->A transversion in position 109 of the inferred cDNA (36R-->S transversion of the processed, N-terminal methionine-lacking CRYGD). The lens protein crystals were X-ray diffracting, and our crystal structure solution at 2.25 A suggests that mutant R36S CRYGD has an unaltered protein fold. In contrast, the observed crystal packing is possible only with the mutant protein molecules that lack the bulky Arg36 side chain. This is the first described case of human cataract caused by crystallization of a protein in the lens. It involves the third known mutation in the CRYGD gene but offers, for the first time, a causative explanation of the phenotype.
Subject(s)
Alleles , Cataract/genetics , Crystallins/genetics , Genetic Linkage , Amino Acid Sequence , Child, Preschool , Crystallins/chemistry , Crystallography, X-Ray , Humans , Lens, Crystalline/pathology , Lens, Crystalline/ultrastructure , Male , Models, Molecular , Molecular Sequence Data , Phenotype , Protein Conformation , Sequence Analysis, DNAABSTRACT
The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.
