MAPPING THE MAB-383C EPITOPE TO ALPHA(2)(187-199) OF THE TORPEDO ACETYLCHOLINE-RECEPTOR ON THE 3-DIMENSIONAL MODEL

Monoclonal. antibody 383C is an anti-acetylcholine receptor antibody w hose binding to the receptor is blocked by alpha-bungarotoxin and by c arbamylcholine. Monoclonal antibody 383C binds to the alpha subunit of the Torpedo acetylcholine (ACh) receptor as well as to its V8-proteas e 20 kDa fragment that possesses the affinity alkylatable Cys192/193. In an epitope scanning experiment spanning the N-terminal 211 amino ac id residues of the alpha subunit, 383C binds uniquely to three overlap ping peptides; alpha(184-196), alpha(187-199) and alpha(190-202). Thes e peptides span a cluster of amino acid residues implicated in the bin ding of acetylcholine, including Cys192/193. To map the location of th ese residues on the three-dimensional model of the ACh receptor, we ha ve employed a combination of X-ray diffraction from oriented complexes of 383C with ACh receptor-enriched membrane vesicles and electron mic roscopy of negatively stained tubular arrays of 383C/receptor complexe s. The X-ray diffraction study finds; extra electron density in the pr esence of 383C centered 35 Angstrom above the synaptic side phosphate head groups. The electron micrographic images display extra stain excl usion from the antibody at a site adjacent to the alpha(2) subunit on the periphery of the rosette clockwise to the alpha(2) vertex. This ma pping localizes several residues of the ACh receptor alpha subunit inv olved in the binding of acetylcholine. Despite these residues being pr esent in both alpha subunits, only the alpha(2) subunit is decorated w ith this monoclonal antibody. (C) 1998 Academic Press.