title: Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6.
abstract: Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.
authors.affiliation: Department of CBRN Defence and Security, Swedish Defence Research Agency, SE-90182 Umea, Sweden;
authors.forename: Anders
authors.initials: A
authors.lastname: Allgardsson
authors.affiliation: Department of Chemistry, Umeå University, SE-90187 Umea, Sweden;
authors.forename: Lotta
authors.initials: L
authors.lastname: Berg
authors.affiliation: Department of CBRN Defence and Security, Swedish Defence Research Agency, SE-90182 Umea, Sweden;
authors.forename: Christine
authors.initials: C
authors.lastname: Akfur
authors.affiliation: SP Processum AB, SE-891 22 Ornskoldsvik, Sweden;
authors.forename: Andreas
authors.initials: A
authors.lastname: Hörnberg
authors.affiliation: Department of Toxicological Enzymology, Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Franz
authors.identifier: 0000-0003-3531-3616
authors.initials: F
authors.lastname: Worek
authors.affiliation: Department of Chemistry, Umeå University, SE-90187 Umea, Sweden; anna.linusson@umu.se freeks@foi.se.
authors.forename: Anna
authors.initials: A
authors.lastname: Linusson
authors.affiliation: Department of CBRN Defence and Security, Swedish Defence Research Agency, SE-90182 Umea, Sweden; anna.linusson@umu.se freeks@foi.se.
authors.forename: Fredrik J
authors.initials: FJ
authors.lastname: Ekström
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry*; D000931:Antidotes / Q000737:chemistry*; D002801:Cholinesterase Reactivators / Q000737:chemistry*; D018360:Crystallography, X-Ray; D007700:Kinetics; D008968:Molecular Conformation; D000067397:Nerve Agents / Q000737:chemistry*; D010091:Oximes / Q000737:chemistry*; D011726:Pyridinium Compounds / Q000737:chemistry*; D012524:Sarin / Q000737:chemistry*
chemical_list: D000931:Antidotes; D002801:Cholinesterase Reactivators; D000067397:Nerve Agents; D010091:Oximes; D011726:Pyridinium Compounds; D012524:Sarin; D000110:Acetylcholinesterase; C022870:asoxime chloride
keywords: acetylcholinesterase; crystallography; density functional theory; nerve agent; reactivation