Mapping the mab 383C epitope to α2(187–199) of the Torpedo acetylcholine receptor on the three-dimensional model

Abstract

Monoclonal antibody 383C is an anti-acetylcholine receptor antibody whose binding to the receptor is blocked by α-bungarotoxin and by carbamylcholine. Monoclonal antibody 383C binds to the α subunit of the Torpedo acetylcholine (ACh) receptor as well as to its V8-protease 20 kDa fragment that possesses the affinity alkylatable Cys192/193. In an epitope scanning experiment spanning the N-terminal 211 amino acid residues of the α subunit, 383C binds uniquely to three overlapping peptides; α(184–196), α(187–199) and α(190–202). These peptides span a cluster of amino acid residues implicated in the binding of acetylcholine, including Cys192/193.To map the location of these residues on the three-dimensional model of the ACh receptor, we have employed a combination of X-ray diffraction from oriented complexes of 383C with ACh receptor-enriched membrane vesicles and electron microscopy of negatively stained tubular arrays of 383C/receptor complexes. The X-ray diffraction study finds extra electron density in the presence of 383C centered 35 Å above the synaptic side phosphate head groups. The electron micrographic images display extra stain exclusion from the antibody at a site adjacent to the α2 subunit on the periphery of the rosette clockwise to the α2 vertex. This mapping localizes several residues of the ACh receptor α subunit involved in the binding of acetylcholine. Despite these residues being present in both α subunits, only the α2 subunit is decorated with this monoclonal antibody.