Crystal Structure of Thioflavin T Bound to the Peripheral Site of Torpedo californica Acetylcholinesterase Reveals How Thioflavin T Acts as a Sensitive Fluorescent Reporter of Ligand Binding to the Acylation Site
- 31 May 2008
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 130 (25), 7856-7861
- https://doi.org/10.1021/ja7109822
Abstract
Acetylcholinesterase plays a key role in cholinergic synaptic transmission by hydrolyzing the neurotransmitter acetylcholine with one of the highest known catalytic rate constants. Hydrolysis occurs in a narrow and deep gorge that contains two sites of ligand binding: A peripheral site, or P-site, near the gorge entrance that contributes to catalytic efficiency both by transiently trapping substrate molecules as they enter the gorge and by allosterically accelerating the transfer of the substrate acyl group to a serine hydroxyl in an acylation site or A-site at the base of the gorge. Thioflavin T is a useful reporter of ligand interactions with the A-site. It binds specifically to the P-site with fluorescence that is enhanced ∼1000-fold over that of unbound thioflavin T, and the enhanced fluorescence is quenched 1.5- to 4-fold when another ligand binds to the A-site in a ternary complex. To clarify the structural basis of this advantageous signal change, we here report the X-ray structure of the complex of thioflavin T with Torpedo californica acetylcholinesterase. The two aromatic rings in thioflavin T are coplanar and are packed snugly parallel to the aromatic side chains of Trp279, Tyr334, and Phe330. Overlays of this structure with the crystal structures of Torpedo californica acetylcholinesterase complexes with either edrophonium or m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone, two small aromatic ligands that bind specifically to the A-site, indicate that the phenyl side chain of Phe330 must rotate to sterically accommodate both thioflavin T and the A-site ligand in the ternary complex. This rotation may allow some relaxation of the strict coplanarity of the aromatic rings in the bound thioflavin T and result in partial quenching of its fluorescence.Keywords
This publication has 44 references indexed in Scilit:
- Structural insights into substrate traffic and inhibition in acetylcholinesteraseThe EMBO Journal, 2006
- Coot: model-building tools for molecular graphicsActa Crystallographica Section D-Biological Crystallography, 2004
- Unmasking Tandem Site Interaction in Human Acetylcholinesterase. Substrate Activation with a Cationic Acetanilide SubstrateBiochemistry, 2003
- Acetylcholinesterase Complexed with Bivalent Ligands Related to Huperzine A: Experimental Evidence for Species-Dependent Protein−Ligand ComplementarityJournal of the American Chemical Society, 2002
- Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo californicaJournal of Molecular Biology, 2000
- Structure of acetylcholinesterase complexed with the nootropic alkaloid, (–)-huperzine ANature Structural & Molecular Biology, 1997
- Acetylcholinesterase inhibition by fasciculin: Crystal structure of the complexCell, 1995
- Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constantsJournal of Applied Crystallography, 1993
- Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase CJournal of Molecular Biology, 1988
- Catalysis by acetylcholinesterase. I. Fluorescent titration with a carbamoylating agentBiochemistry, 1971