Gerlits Oksana, Blakeley Matthew P, Keen David A, Radić Zoran, Kovalevsky Andrey
Department of Natural Sciences, Tennessee Wesleyan University, Athens, TN, 37303, USA.
Large Scale Structures Group, Institut Laue-Langevin, 38000, Grenoble, France.
Curr Res Struct Biol. 2021 Sep 6;3:206-215. doi: 10.1016/j.crstbi.2021.08.003. eCollection 2021.
Acetylcholinesterase (AChE) catalyzes hydrolysis of acetylcholine thereby terminating cholinergic nerve impulses for efficient neurotransmission. Human AChE (hAChE) is a target of nerve agent and pesticide organophosphorus compounds that covalently attach to the catalytic Ser203 residue. Reactivation of inhibited hAChE can be achieved with nucleophilic antidotes, such as oximes. Understanding structural and electrostatic (i.e. protonation states) determinants of the catalytic and reactivation processes is crucial to improve design of oxime reactivators. Here we report X-ray structures of hAChE conjugated with a reversible covalent inhibitor 4K-TMA (4K-TMA:hAChE) at 2.8 Å resolution and of 4K-TMA:hAChE conjugate with oxime reactivator methoxime, MMB4 (4K-TMA:hAChE:MMB4) at 2.6 Å resolution, both at physiologically relevant room temperature, as well as cryo-crystallographic structure of 4K-TMA:hAChE at 2.4 Å resolution. 4K-TMA acts as a substrate analogue reacting with the hydroxyl of Ser203 and generating a reversible tetrahedral hemiketal intermediate that closely resembles the first tetrahedral intermediate state during hAChE-catalyzed acetylcholine hydrolysis. Structural comparisons of room temperature with cryo-crystallographic structures of 4K-TMA:hAChE and published mAChE complexes with 4K-TMA, as well as the effect of MMB4 binding to the peripheral anionic site (PAS) of the 4K-TMA:hAChE complex, revealed only discrete, minor differences. The active center geometry of AChE, already highly evolved for the efficient catalysis, was thus indicative of only minor conformational adjustments to accommodate the tetrahedral intermediate in the hydrolysis of the neurotransmitter acetylcholine (ACh). To map protonation states in the hAChE active site gorge we collected 3.5 Å neutron diffraction data paving the way for obtaining higher resolution datasets that will be needed to determine locations of individual hydrogen atoms.
乙酰胆碱酯酶（AChE）催化乙酰胆碱水解，从而终止胆碱能神经冲动以实现高效的神经传递。人乙酰胆碱酯酶（hAChE）是神经毒剂和有机磷农药化合物的作用靶点，这些化合物会与催化性丝氨酸203残基共价结合。使用亲核解毒剂（如肟类化合物）可实现被抑制的hAChE的重新激活。了解催化和重新激活过程的结构和静电（即质子化状态）决定因素对于改进肟类重新激活剂的设计至关重要。在此，我们报告了在生理相关的室温下，分辨率为2.8 Å的hAChE与可逆共价抑制剂4K-TMA（4K-TMA:hAChE）的X射线结构，以及分辨率为2.6 Å的4K-TMA:hAChE与肟类重新激活剂甲氧肟（MMB4）的复合物（4K-TMA:hAChE:MMB4）的X射线结构，还有分辨率为2.4 Å的4K-TMA:hAChE的低温晶体结构。4K-TMA作为底物类似物与丝氨酸203的羟基反应，生成可逆的四面体半缩酮中间体，该中间体与hAChE催化乙酰胆碱水解过程中的第一个四面体中间态非常相似。对4K-TMA:hAChE的室温结构与低温晶体结构以及已发表的mAChE与4K-TMA复合物进行结构比较，以及MMB4与4K-TMA:hAChE复合物外周阴离子位点（PAS）结合的影响，结果仅显示出离散的、微小的差异。AChE的活性中心几何结构已经为高效催化高度进化，因此仅表明在适应神经递质乙酰胆碱（ACh）水解中的四面体中间体时发生了微小的构象调整。为了绘制hAChE活性位点峡谷中的质子化状态图，我们收集了3.5 Å的中子衍射数据，为获取确定单个氢原子位置所需的更高分辨率数据集铺平了道路。
