Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase.
Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, and quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-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 close interaction was confirmed by photoaffinity labelling by the photosensitive probe 3H-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label 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 of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.
Harel
M
M
Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
Schalk
I
I
Ehret-Sabatier
L
L
Bouet
F
F
Goeldner
M
M
Hirth
C
C
Axelsen
P H
PH
Silman
I
I
Sussman
J L
JL
D000109:Acetylcholine / Q000378:metabolism; D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray / Q000379:methods; D003650:Decamethonium Compounds / Q000378:metabolism; D004491:Edrophonium / Q000378:metabolism; D006860:Hydrogen Bonding; D008024:Ligands; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D013619:Tacrine / Q000378:metabolism; D014101:Torpedo
D000345:Affinity Labels; D003650:Decamethonium Compounds; D008024:Ligands; D010446:Peptide Fragments; D000644:Quaternary Ammonium Compounds; D013619:Tacrine; D004491:Edrophonium; C033019:decamethonium; D000110:Acetylcholinesterase; C027917:tetramethylammonium; D000109:Acetylcholine