Room temperature crystallography of human acetylcholinesterase bound to a substrate analogue 4K-TMA: Towards a neutron structure.
Room temperature crystallography of human acetylcholinesterase bound to a substrate analogue 4K-TMA: Towards a neutron structure.
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与底物类似物4K-TMA结合的人乙酰胆碱酯酶的室温晶体学：朝向中子结构。
DOI：
10.1016/j.crstbi.2021.08.003
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发表时间：
2021
影响因子：
2.8
通讯作者：
Kovalevsky A
中科院分区：
文献类型：
--
作者：
Gerlits O;Blakeley MP;Keen DA;Radić Z;Kovalevsky A
乙酰胆碱酯酶(AChE)催化乙酰胆碱的水解，从而终止胆碱能神经冲动，以实现有效的神经传递。人类乙酰胆碱酯酶(HAChE)是神经毒剂和农药有机磷化合物的靶标，这些化合物共价结合在催化的Ser203残基上。被抑制的HAChE可以用亲核解毒剂，如肟类实现重新激活。了解催化和再活化过程的结构和静电(即质子化状态)决定因素对于改进肟类复活剂的设计是至关重要的。本文报道了4K-TMA(4K-TMA：HAChE)与可逆共价抑制剂4K-TMA(4K-TMA：HAChE)在2.8x？分辨率下和4K-TMA：HAChE与肟复活剂甲氧磷定MMB4(4K-TMA：HAChE：MMB4)在2.6？分辨率下的X-射线结构，以及4K-TMA：HAChE在2.4？分辨率下的低温晶体结构。4K-TMA作为底物类似物，与Ser203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催化的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和已发表的4K-TMA-HAChE络合物的室温结构与冷晶学结构进行了结构比较，以及MMB4与4K-TMA：HAChE络合物的外周阴离子中心(PAS)结合的影响，仅显示了离散的、微小的差异。乙酰胆碱酯酶的活性中心几何构型已经为高效催化而高度进化，因此表明只有微小的构象调整才能适应神经递质乙酰胆碱(ACh)的水解过程中的四面体中间体。为了绘制HACH活性中心峡谷中的质子化状态图，我们收集了3.5兆ä中子衍射数据，为获得确定单个氢原子位置所需的更高分辨率的数据集铺平了道路。4K-TMA的共价结合模拟了HAChE催化中的四面体中间体。在RT-X-射线结构中没有观察到4K-TMA与外周阴离子中心的结合。在4K-TMA：HAChE络合物中，肟类复活剂MMB4与外周阴离子结合。3.5ä分辨率中子衍射数据为解决质子化状态铺平了道路。
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. Covalent binding of 4K-TMA mimics the tetrahedral intermediate in hAChE catalysis. No binding of 4K-TMA to the peripheral anionic site is observed in RT X-ray structure. Oxime reactivator MMB4 binds to the peripheral anionic site in 4K-TMA:hAChE complex. 3.5 Å resolution neutron diffraction data pave the way to resolve protonation states.
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影响因子：
14.9
作者：
wwPDB consortium
通讯作者：
wwPDB consortium
影响因子：
4.8
作者：
Cochran, Rory;Kalisiak, Jaroslaw;Taylor, Palmer
通讯作者：
Taylor, Palmer
影响因子：
4.8
作者：
Bourne, Yves;Radic, Zoran;Marchot, Pascale
通讯作者：
Marchot, Pascale
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
6.1
作者：
Coates, Leighton;Robertson, Lee
通讯作者：
Robertson, Lee