Molecular and structural basis of the specificity of a neutralizing acetylcholine receptor-mimicking antibody, using combined mutational and molecular modeling analyses

The antagonist activity of short-chain toxins from snake venoms toward the nicotinic acetylcholine receptor (nAChR) is neutralized upon binding to a t oxin-specific monoclonal antibody called M alpha 2-3 (1). To establish the molecular basis of this specificity, we predicted from both mutational anal yses and docking procedures the structure of the M alpha 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 complementar ity 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 selec tion procedure yielded three models based on docking calculations. The sele cted models predicted the proximity of Tyr(49) and/or Tyr(50) in the antibo dy to Lys(47) in the toxin. This was experimentally confirmed using another round of double-mutation cycles. The two models finally selected were subm itted to energy minimization in a CHARMM22 force field, and were characteri zed by a root mean square deviation of 7.0 +/- 2.9 Angstrom. Both models di splay most features of antibody-antigen structures. Since M alpha 2-3 also partially mimics some binding properties of nAChR, these structural feature s not only explain its fine specificity of recognition, but may also furthe r clarify how toxins bind to nAChR.