Interaction between a Fab fragment against gp41 of human immunodeficiency virus 1 and its peptide epitope: characterization using a peptide epitope library and molecular modeling

Abstract

The molecular interaction of the Fab fragment of the human monoclonal antibody 3D6, directed against the transmembrane protein gp41 of human immunodeficiency virus (HIV) 1, with its peptide epitope is characterized by a panel of overlapping peptides, a peptide epitope library and molecular modeling techniques. The sequence CSGKLICTTAVPW, corresponding to amino acids 605-617 of gp41, was identified as the best binding peptide (KD = 1 x 10(-8) mol/l). This peptide served as a starting point to prepare a cellulose-bound peptide epitope library in which each residue of the epitope is substituted by all L- and D-amino acids, resulting in 494 epitope peptide variants which were subsequently analyzed for binding 3D6. The library was synthesized to identify residues critical for binding and to obtain information about the molecular environment of the epitope peptide bound to 3D6. Both cysteine residues, as well as isoleucine 6, threonine 8 and proline 12, of the epitope were highly sensitive to substitution. Using the data obtained from the epitope characterization, as well as a low-resolution electron density map of a 3D6 Fab-peptide complex, a 3-D model of the Fab-peptide complex was generated by molecular modeling. The modeling experiments predict binding of the peptide, which is cyclized via the two cysteine residues, to a pocket formed dominantly by the hypervariable loops complementarity determining regions CDR3L, CDR2H and CDR3H.

Engineering Regulable Escherichia coliβ-Galactosidases as Biosensors for Anti-HIV Antibody Detection in Human Sera

“…1A). Two overlapping HIV epitopes of the env-encoded gp41 glycoprotein, namely P1 (16) and P2 (17,18), contained in five different sized peptides (Fig. 1B) were inserted either at position 278 or position 795, both located in permissive solvent-exposed loops of the assembled enzyme tetramer.…”

“…3B). Noticeably, a model of the anti-HIV 3D6 Fab fragment bound to the P2 epitope predicts the cyclization of this epitope via the two cysteine residues that are critical for antibody-epitope interaction (17). Because in the cytoplasm of E. coli proteins are in a reduced state, the disulfide bond formation in the P2 epitope-containing recombinant proteins is not favored, and this fact could prevent correct B-cell epitope presentation.…”

“…By a detailed characterization of the resulting engineered enzymes, we show here that some of them containing P1 (16) and P2 (17,18) immunodominant epitopes from gp41 are highly antigenic and responsive to binding of anti-epitope antibodies, reaching reactivation factors higher than 250%. In addition, we demonstrate that the regulable enzymes specifi-cally respond to anti-HIV-1 antibodies in serum of infected patients, proving the high performance and robustness of these biosensors in complex samples and their usefulness in real diagnostic situations.…”

Engineering of Escherichia coli β‐galactosidase for solvent display of a functional scFv antibody fragment

“…The 21‐mer linear peptide A21 (YTASARGDLAHLTTTHARHLP) reproduces a segment of the G‐H loop from FMDV VP1 protein, the antigenic site A, which includes the antibody SD6 B‐cell epitope [13]. The 13‐mer control peptide P2 (CSGKLICTTAVPW) reproduces an immune‐relevant B‐cell epitope from gp41 of HIV [18]. Binding of PANlacZSD6 to peptides was tested in solid phase by sandwich ELISA as follows.…”

Selection of antibody probes to correlate protein sequence domains with their structural distribution

“…is a simple, flexible technique for simultaneous parallel chemical synthesis on porous membrane supports such as filter paper, allowing rapid automated distribution of amino acids and low-cost production of large numbers of peptides of use for systematic epitope analysis. The success of this method is reflected in its use in epitope mapping, peptide size analysis, analogue-scan, single Ala substitution analysis~Frank, 1992; Höhne et al, 1993;Commandeur et al, 1994;Reusch et al, 1994;Martens et al, 1995;Stigler et al, 1995;Reineke et al, 1996!, analysis of combinatorial libraries for substrate recognition motifs of cAMP-and cGMP-dependent kinases, identification of metal-or nucleic acid-binding peptides~Kramer et Malin et al, 1995;Tegge & Frank, 1995! and the study of the molecular basis of the binding promiscuity displayed by a monoclonal antibody~Kramer et al, 1997!.…”