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

Michal Harel, Leilani K. Sonoda, Israel Silman, Joel L. Sussman, Terrone L. Rosenberry

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)7856-7861
Number of pages6
JournalJournal of the American Chemical Society
Volume130
Issue number25
DOIs
StatePublished - Jun 25 2008

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Torpedo
Acylation
Acetylcholinesterase
Crystal structure
Ligands
Fluorescence
Edrophonium
Substrates
Quenching
Hydrolysis
Rate constants
Synaptic Transmission
Hydroxyl Radical
Serine
Cholinergic Agents
Acetylcholine
Neurotransmitter Agents
thioflavin T
X rays
Binding Sites

ASJC Scopus subject areas

  • Chemistry(all)

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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. / Harel, Michal; Sonoda, Leilani K.; Silman, Israel; Sussman, Joel L.; Rosenberry, Terrone L.

In: Journal of the American Chemical Society, Vol. 130, No. 25, 25.06.2008, p. 7856-7861.

Research output: Contribution to journalArticle

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