Structural basis of HIV-1 and HIV-2 protease
by a monoclonal antibody.
P. Řezáčová1, J. Lescar2, J. Brynda1, M. Fábry1, M. Hořejší1, G.A. Bentley3 and J. Sedláček1
1Dep. of Gene Manipulation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6, Czech Republic
2 School of Biological Sciences, Nanyang Walk, Blk 5, Level 3, Singapore 637616
3 Unité d’Immunologie Structurale, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, France
Since the demonstration that the Human Immunodeficiency Virus protease (HIV PR) is essential in the viral life cycle , this enzyme has become one of the primary targets for antiviral drug design. With the objective of probing the structural stability of HIV PR and the eventual design of potential inhibitors directed to regions other than the active site, we have examined the effects of anti-HIV-1 PR monoclonal antibodies (mAbs) on the catalytic activity of the protease [2, 3]. Monoclonal antibody mAb1696, although raised against the HIV-1 PR, inhibits the catalytic activity of both the HIV-1 and HIV-2 enzymes with inhibition constants in nanomolar range  and cross-reacts with peptides comprising the N‑terminus of the HIV protease (residues 1 to 7). The N‑terminal region is essential for dimerization of monomers and thus forming the active HIV PR dimer.
To investigate further the mechanism of HIV PR inhibition by mAb1696, a recombinant single-chain Fv fragment (scFv) that contains haeavy and light chain variable domains of mAb1696 joined by a flexible linker have been prepared and used for crystallographic studies . Although the crystallization of scFv1696 complexed with the HIV proteases has not yet been successful, the formation of these complexes has been demonstrated in solution. As an alternative approach to studying the antigenic recognition by the mAb 1696 at the structural level, we have crystallized complexes of scFv1696 with the respective epitope peptide fragments of the HIV protease. The three‑dimensional structure of the complex formed between scFv1696 and epitope peptide PQITLWQRR (corresponding to the N‑terminus of HIV-1 PR) has been solved and refined at 2.70 Å resolution . The structure of the complex formed between scFv1696 and epitope peptide PQFSLWKR (corresponding to the N‑terminus of HIV-2 PR) has been solved and refined at 1.88 Å resolution.
Interactions of peptides with scFv1696 have been analyzed and compared in both structures. On the basis of the interactions seen in the complex, the cross-reactivity between mAb1696 and the HIV-1 and HIV-2 protease and their N‑terminal peptides can be explained. The broad reactivity between mAb 1696 on one side and the HIV-1 PR and HIV-2 PR derives from the invariance or conservation of the first six N-terminal residues of the two strains. Most of these residues are deeply buried in the antibody-binding groove and establish extensive contacts.
Using the peptide as a guide, a docking complex of a whole protease monomer was generated, which suggests that mAb 1696 inhibits the HIV PR by favouring the dissociation of the active homodimer. A dissociative mechanism of protease inhibition by 1696 is consistent with the stoichiometry of the inhibition complex, as derived from the inhibition kinetic studies .
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