In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (M␣2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (1). Further confirming this mimicry, we show that (i) like AChR, M␣2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106 -122 of the ␣-chain of AChR shares 66% primary structure identity with complementarity-determining regions of M␣2-3. Also, a mutational analysis of erabutoxin a reveals that the epitope recognized by M␣2-3 consists of 10 residues, distributed within the three toxin loops. Eight of these residues also belong to the 10-residue epitope recognized by AChR, a result that offers an explanation as to the functional similarities between the receptor and the antibody. Strikingly, however, most of the residues common to the two epitopes contribute differentially to the energetic formation of the antibody-toxin and the receptor-toxin complexes. Together, the data suggest that the mimicry between AChR and M␣2-3 is partial only.
M␣2-3 is a monoclonal antibody that partially mimics the nicotinic acetylcholine receptor (AChR). Its threedimensional structure has been previously predicted by molecular modeling, suggesting that 29 complementarity determining region (CDR) residues and 2 framework residues are exposed to solvent. To identify the antibody residues that bind to the antigen, i.e. snake toxin that binds specifically to AChR, we (i) produced the scFv form of M␣2-3 fused to alkaline phosphatase, in the periplasmic space of Escherichia coli; (ii) submitted approximately 75% of exposed residues of the fused scFv to individual or combined mutations, and (iii) identified the residues whose mutations affect scFv binding to the toxin, using a sensitive enzyme-linked immunosorbent assay. 11 critical residues were identified, including 8 heavy chain residues, 2 framework residues, and 1 light chain residue. They cover a surface of approximately 800 Å 2 , with a subset of most critical residues (VHD31, VHY32, and VHG101) and several aromatic residues. This functional architecture not only constitutes a plausible complementary binding surface for the snake toxin but also offers a structural basis to ultimately understand the capacity of the antibody to partially mimic AChR.
The antagonist activity of short-chain toxins from snake venoms toward the nicotinic acetylcholine receptor (nAChR) is neutralized upon binding to a toxin-specific monoclonal antibody called M␣2-3 (1). To establish the molecular basis of this specificity, we predicted from both mutational analyses and docking procedures the structure of the M␣2-3-toxin complex. From knowledge of the functional paratope and epitope, and using a double-mutation cycle procedure, we gathered evidence that Asp 31 in complementarity determining region 1H is close to, and perhaps interacts with, Arg 33 in the antigen. The use of this pair of proximate residues during the selection procedure yielded three models based on docking calculations. The selected models predicted the proximity of Tyr 49 and/or Tyr 50 in the antibody to Lys 47 in the toxin. This was experimentally confirmed using another round of double-mutation cycles. The two models finally selected were submitted to energy minimization in a CHARMM22 force field, and were characterized by a root mean square deviation of 7.0 ؎ 2.9 Å. Both models display most features of antibody-antigen structures. Since M␣2-3 also partially mimics some binding properties of nAChR, these structural features not only explain its fine specificity of recognition, but may also further clarify how toxins bind to nAChR.It is of particular interest in the case of toxic antigens to understand the structural characteristics and molecular events underlying the binding properties of neutralizing antibodies. M␣2-3 is a murine monoclonal antibody (IgG2a/ isotype), which binds with high affinity to potent antagonists of the nicotinic acetylcholine receptor (nAChR) 1 (1). Curaremimetic short-chain toxins from Elapidae and Hydrophiidae snake venoms form a family of small proteins comprising 60 -62 amino acid residues and 4 disulfides. They bind to nAChR with high affinities, their apparent equilibrium dissociation constants ranging between 10 Ϫ10 and 10 Ϫ12 M (2). Unlike nAChR, M␣2-3 does not bind long-chain neurotoxins, a second family of structurally related snake antagonists. Therefore, the main goal of this study was to establish how M␣2-3 specifically and exclusively recognizes short-chain neurotoxins. Additionally, we had previously shown that M␣2-3 partially mimics nAChR (3). Among the properties shared with nAChR, M␣2-3 induces antinAChR antibodies that block binding of the antagonists to nAChR, and both the antibody and the receptor recognize highly similar topographies on the surface of the antagonist molecules. Therefore, clarification of how a short-chain toxin binds to the antibody may also help to understand how it binds to nAChR.Determination of the three-dimensional structure of an antibody, free or complexed to its antigen, is presently based on x-ray crystallographic analyses (4, 5). During the past two decades, the number of crystal structures of antibody fragments has been rising, and to date over 60 structures have been solved. In contrast, the number of elucidated three-dimensional struct...
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