UC San Diego

The acetylcholine binding protein (AChBP) is a structural and functional surrogate of the nicotinic acetylcholine receptor (nAChR) ligand binding domain (LBD). AChBP is a natural truncation of the nAChR. The soluble protein is secreted into the cholinergic synapse of mollusks as a primitive synaptic modulator. The solubility of AChBP is amenable to fluorescence spectroscopy and X-ray crystallography and provides an opportunity to investigate ligand related conformational changes and structural determinants of ligand binding. Material for studying Lymnaea and Aplysia AChBP was obtained by chemically synthesizing genes (cDNA) encoding for the proteins. A mammalian expression system was devised in combination with a FLAG-antibody immunopurification protocol. A single FLAG tag on the N-terminus was optimal for expression of both proteins. Ligand binding of most agonists and antagonists resulted in a substantial change in intrinsic tryptophan fluorescence. Kinetic measurements by stopped- flow techniques showed rapid rates of association and dissociation for agonists and slow rates for the a- neurotoxins. Equilibrium dissociation constants for ligands with rates beyond the detection of stopped-flow were measured in a fluorescent plate reader at equilibrium. AChBP from Aplysia californica was shown to have five orders of magnitude higher affinity for a- conotoxin ImI. Crystal structures were determined for agonists and antagonists bound to AChBP. Antagonists included a-conotoxin ImI, a-cobratoxin, and methyllycaconitine. Antagonist binding was compared to that of the agonists epibatidine and lobeline. Ten side chains were associated with all bound ligands delineating the competitive hydrophobic binding pocket. Antagonists occupied two additional non-overlapping binding surfaces; one on the apical side of the C loop the another on the 'membrane' side under the C loop. Changes in conformational states of AChBP were investigated by comparing a crystal structure of apo Aplysia AChBP with those of antagonists and agonists. The C loop undergoes a large conformational change to cap the binding pocket when agonists are bound. Antagonists extend the C loop radially up to 11 Å when compared to agonists. Knowledge of structural determinants for binding selective ligands and associated conformational changes should facilitate rational drug design and the understanding of nAChR function