NMR SOLUTION STRUCTURE OF AN ALPHA-BUNGAROTOXIN NICOTINIC RECEPTOR PEPTIDE COMPLEX

We report the two-dimensional nuclear magnetic resonance (NMR) charact erization of the stoichiometric complex formed between the snake venom -derived long alpha-neurotoxin, alpha-bungarotoxin (BGTX), and a synth etic dodecapeptide (alpha185-196) corresponding to a functionally impo rtant region on the alpha-subunit of the nicotinic acetylcholine recep tor (nAChR) obtained from Torpedo californica electric organ tissue. B GTX has been widely used as the classic nicotinic competitive antagoni st for the skeletal muscle type of nAChR which is found in the avian, amphibian, and mammalian neuromuscular junction. The receptor dodecape ptide (alpha185-196) binds BGTX with micromolar affinity and has been shown to represent the major determinant of BGTX binding to the isolat ed alpha-subunit. Previous studies involving covalent modification of the native nAChR from Torpedo membranes with a variety of affinity rea gents indicate that several residues contained within the dodecapeptid e sequence (namely, Tyr-190, Cys-192, and Cys-193) apparently contribu te directly to the formation of the cholinergic ligand binding site. T he NMR-derived solution structure of the BGTX/receptor peptide complex defines a relatively extended conformation for a major segment of the ''bound'' dodecapeptide. These structural studies also reveal a previ ously unpredicted receptor binding cleft within BGTX and suggest that BGTX undergoes a conformational change upon peptide binding. If, as we hypothesize, the identified intermolecular contacts in the BGTX/recep tor peptide complex describe a portion of the contact zone between BGT X and native receptor, then the structural data would suggest that alp ha-subunit residues 186-190 are on the extracellular surface of the re ceptor.