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
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
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DOI：
10.1021/ja7109822
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发表时间：
2008-06-25
影响因子：
15
通讯作者：
Rosenberry, Terrone L.
中科院分区：
文献类型：
--
作者：
Harel, Michal;Sonoda, Leilani K.;Rosenberry, Terrone L.
乙酰胆碱酯酶通过以已知最高催化速率常数之一水解神经递质乙酰胆碱，在胆碱能突触传递中发挥关键作用。水解发生在狭窄而深的峡谷中，该峡谷包含两个配体结合位点：靠近峡谷入口的外围位点或 P 位点，它通过在底物分子进入峡谷时瞬时捕获它们和通过变构加速底物酰基向峡谷底部的酰化位点或 A 位点中的丝氨酸羟基的转移来提高催化效率。硫磺素 T 是配体与 A 位相互作用的有用报告分子。它与 P 位点特异性结合，其荧光强度比未结合的硫黄素 T 增强了 1000 倍，并且当另一个配体与三元复合物中的 A 位点结合时，增强的荧光会猝灭 1.5 至 4 倍。为了阐明这种有利信号变化的结构基础，我们在此报告了硫黄素 T 与加州鱼雷乙酰胆碱酯酶复合物的 X 射线结构。硫代黄素 T 中的两个芳香环是共面的，并且与 Trp279、Tyr334 和 Phe330 的芳香侧链紧密平行地排列。该结构与鱼雷鱼乙酰胆碱酯酶复合物的晶体结构与依酚鎓或间-(N,N,N-三甲基铵)-2,2,2-三氟苯乙酮（两个与A位点特异性结合的小芳香族配体）的晶体结构重叠，表明Phe330的苯基侧链必须旋转以在空间上容纳三元复合物中的硫黄素T和A位配体。这种旋转可能会使结合的硫代黄素 T 中芳环的严格共面性有所松弛，并导致其荧光部分猝灭。
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 similar to 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.