Backbone Conformation Shifts in X-ray Structures of Human Acetylcholinesterase upon Covalent Organophosphate Inhibition

Stephanie Luedtke,Bianca Pomar,Yunshen Li,Zoran Radić,Celine Bojo,Emilio Luna

doi:10.3390/cryst11111270

Abstract

Conformations of Cα backbones in X-ray structures of most organophosphate (OP)-inhibited human acetylcholinesterases (hAChEs) have been previously shown to be similar to that of the native hAChE. One of the exceptions is the structure of the diethylphosphoryl-hAChE conjugate, where stabilization of a large ethoxy group into the acyl pocket (AP) of hAChE-triggered notable loop distortions and consequential dissociation of the hAChE homodimer. Recently, six X-ray structures of hAChE conjugated with large OP nerve agents of the A-type, Novichoks, have been deposited to PDB. In this study we analyzed backbone conformation shifts in those structures, as well as in OP-hAChE conjugates formed by Paraoxon, Soman, Tabun, and VX. A Java-based pairwise alpha carbon comparison tool (PACCT 3) was used for analysis. Surprisingly, despite the snug fit of large substituents on phosphorus, inside Novichok-conjugated hAChEs only minor conformational changes were detected in their backbones. Small magnitudes of observed changes were due to a 1.2–2.4 Å shift of the entire conjugated OP away from the AP. It thus appears that the small AP of AChEs can accommodate, without distortion, substituents of the size of ethoxy or butyryl groups, provided that conjugated OP is “pulled” away from the AP. This observation has practical consequences in the structure-based design of nucleophilic reactivation antidotes as well as in the definition of the AChE specificity that relies on the size of its AP.

Highlights

Triesters of phosphoric acid, diesters of phosphonic acid as well as phosphoramidic acid esters are commonly known as organophosphate (OP) inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7) an essential hydrolytic enzyme in cholinergic neurotransmission of vertebrates [1]
Some of the most toxic substances made by man are OPs, such as nerve agent poisons VX and Novichok OPs, compounds from this class are frequently used as pesticides
Distortion of the hAChE backbone upon conjugation of diethylphosphate of POX, an active inhibitory form of the pesticide parathion, to the Ser203 has been well documented earlier based on RMSD overlay and Rapido-server analysis [13]

Summary

Introduction

Diesters of phosphonic acid as well as phosphoramidic acid esters are commonly known as organophosphate (OP) inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7) an essential hydrolytic enzyme in cholinergic neurotransmission of vertebrates [1]. General susceptibility to hydrolytic degradation allows non-volatile OP pesticides to convert to non-toxic products within days of their application in the field. Nerve agent warfare OPs, on the other hand, are both more stable (VX, Tabun, Novichoks) [4] and much more volatile [1] (sarin, soman) rendering them extremely hazardous. Some and much more volatile [1] (sarin, soman) rendering them extremely hazardous. Some of the OP pesticides such as parathion, chlorpyrifos, or malathion are applied in their low toxicity phosphorothioate (P=S), forms that oxidative enzymes in of insects convert to much more toxic OP “oxons” (P=O). Both pesticide and nerve agent OPs owe their exquisite toxicity to their uncharged chemical nature, which allows them to of the OPtraverse pesticides such as parathion, chlorpyrifos, or malathion are applied in their low quickly biological membranes (such as skin or alveoli), enter blood and penetrate toxicity phosphorothioate (P=S), forms that oxidative enzymes in insects to much both peripheral and central nervous systems, reaching their targetconvert

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