Abstract
With the increase in our understanding of its structure and enzymatic mechanism, HIV-1 integrase (IN) has become a promising target for designing drugs to treat patients with AIDS. To investigate the structure and function of IN, a panel of monoclonal antibodies (mAbs) directed against HIV-1 IN was raised and characterized previously in this laboratory. Among them, mAbs17, -4, and -33 were found to inhibit IN activity in vitro. In this study, we investigated the interaction of N-terminal-specific mAb17 and its isolated Fab fragment with full-length HIV-1 IN(1-288) and its isolated N-terminal, Zn(2+)-binding domain IN(1-49). Our results show that binding of Zn(2+) to IN(1-49) stabilizes the mAb17-IN complex and that dimer dissociation is not required for binding of the Fab. To identify the epitope recognized by mAb17, we developed a protein footprinting technique based on controlled proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Binding was mapped to a region within amino acids Asp(25)-Glu(35). This peptide corresponds to the end of a helix-turn-helix motif in the IN(1-55) NMR structure and contributes to the dimerization of the N-terminal domain. Antibody binding also appears to destabilize the N-terminal helix in this domain. A molecular model of the [IN(1-49)](2).(Fab)(1) complex shows Fab binding across the dimer protein and suggests a potential target for drug design. These data also suggest that mAb17 inhibits integrase activity by blocking critical protein-protein interactions and/or by distorting the orientation of the N-terminal alpha-helix. The relevance of our results to an understanding of IN function is discussed.
Highlights
Monoclonal Antibody 17 and Its Isolated Fab Fragment Inhibit IN Activity—Our initial human immunodeficiency virus type-1 (HIV-1) IN activity assays indicated that several monoclonal antibodies that we had isolated inhibited HIV-1 IN activity in vitro
To determine if this inhibition was due to cross-linking of IN via bivalence of the monoclonal antibodies (mAbs), we isolated its monovalent Fab derivative, and we tested its effect on the HIV-1 IN activity
In this report we have analyzed the molecular interaction between HIV-1 IN and a monoclonal antibody that was known to bind to its N-terminal domain
Summary
Expression and Purification of HIV-1 IN and Derivatives—The construction of plasmid pET29b encoding wild type HIV-1 IN(8) and mutants IN-F185KC2805 (8) and IN-3CS (C56S, C65S, and C280S) (10) has been reported previously. The construction of plasmid pET28b that included a gene encoding the N-terminal domain fragment (amino acids 1– 49) with a six-histidine tag and a thrombin cleavage site on the N-terminal end of the protein was described in a previous report (8). This plasmid was expressed in E. coli BL21(DE3) cells. This antibody has an H2 loop one amino acid longer than the mAb17 H2 loop For this loop we used the 2.7-Å crystal structure of the anti-digoxin antibody 40 –50 in PDB entry 1IBG, chain H (39). The lowest energy structure was accepted and is presented below
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