Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures ofcomplexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, the quaternary nitrogen of the ligand interacts with the indole of Trp-84, and Its m-hydroxyl displays bilbrcated hydrogen bonding to two members of the catalytic triad, Ser-200 and Hls-440. In a complex with tacrine, the acridine Is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium Is oriented along the narrow gorge leading to the active site; one quaternary group Is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This dose Interaction was confirmed by photoaffnity labeling by the photosensitive probe 3H-labeled p-(N,Ndimethylamlno)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active dte. Labeling ofTrp-279 was also observed. One mole oflabel is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role ofaromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.

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Structural changes occurring upon desensiti- were labeled in a roughly identical manner in both resting and desensitized conformations, the labeling of Tyr-93 and Trp-149 increased up to 6-fold in the desensitized state. Tyr-93 and Trp-149 belong to separate regions containing strictly conserved "canonical" amino acids, common to all nicotinic, y-aminobutyrate, and glycine receptor subunits. These regions are thus likely to play a critical role in the regulation of ligand-gated ion channels.Recombinant DNA technology has led to the identification of a large collection of amino acid sequences from several members of the superfamily of ligand-gated ion channels, such as the nicotinic acetylcholine receptor (AcChoR) from electric organ, muscle, and brain, as well as the glycine, 'y-aminobutyrate (GABA), and glutamate receptors (reviewed in refs. 1-15). Structural and functional evidence supports the view that these allosteric membrane proteins (4, 5) are heteropentameric oligomers made up of at least two different categories of subunits, their distinctive pharmacological and physiological properties being associated with a defined subunit composition (6-9, 37, 38). Yet, in the absence of x-ray crystallographic data, little is known about the actual three-dimensional architecture of these oligomers and about the detailed structural mechanisms of the allosteric transitions that mediate fast and slow regulation of ion channel opening.One of the best characterized members of this family is the peripheral nicotinic AcChoR receptor from Torpedo electric organ (4,5,(10)(11)(12). It is an a2,83y oligomer of 300 kDa that contains two acetylcholine (AcCho) binding sites with distinct pharmacological properties and several allosteric sites for pharmacological agents referred to as noncompetitive blockers (4,5). New insights about the tertiary structure and amino acid composition of its binding sites for cholinergic ligands were recently obtained with the photoaffinity ligand p-(N,Ndimethyl)aminobenzenediazonium fluoroborate (DDF) (13)(14)(15). This compound, which behaves in the dark as a reversible competitive antagonist, covalently attaches to the cholinergic binding sites with a 1:1 stoichiometry upon photoactivation by energy transfer from the protein (13). The amino acids labeled by DDF belong to three distinct peptide segments of the large amino-terminal hydrophilic domain of the a subunit, which include Tyr-93, 15), and the two cysteines, 192 and 193, initially identified (16) with a maleimido derivative. Furthermore, DDF also reacts, though to a smaller extent, with the ,B, y, and 8 subunits (13), suggesting that they may each participate in one of the two binding areas in conjunction with one a subunit (17-20) and thereby account for the distinct binding specificity of the two a subunits (21,22).The present work aims at the analysis, at the amino acid level, of the structural reorganizations that occur in the cholinergic ligand-binding domains upon desensitization, a slow reaction (100 msec to 1 min) observ...