Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects

Abstract

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the RP enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an SP-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away. Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the RP enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an SP-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away. Acetylcholinesterase (EC 3.1.1.7; AChE), a key enzyme of cholinergic neurotransmission, is encoded by a single gene in vertebrates, yet in tissues it exists in diverse molecular forms (1Schumacher M. Camp S. Maulet Y. Newton M. MacPhee-Quigley K. Taylor S.S. Friedmann T. Taylor P. Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence.Nature. 1986; 319: 407-409Crossref PubMed Scopus (308) Google Scholar, 2Maulet Y. Camp S. Gibney G. Rachinsky T.L. Ekström T.J. Taylor P. Single gene encodes glycophospholipid-anchored and asymmetric acetylcholinesterase forms: Alternative coding exons contain inverted repeat sequences.Neuron. 1990; 4: 289-301Abstract Full Text PDF PubMed Scopus (72) Google Scholar). AChE is found in different quaternary associations including monomers, homodimers covalently (-S-S-) linked via C-terminal cysteines, homotetramers (two reversibly associated dimers), and clusters of up to three tetramers covalently attached to distinct structural subunits (3Taylor P. The cholinesterases.J. Biol. Chem. 1991; 266: 4025-4028Abstract Full Text PDF PubMed Google Scholar). A likely evolutionary advantage for the clustering of large numbers of catalytic centers of this highly efficient biological catalyst, in neurons and muscle, is to achieve precise spatiotemporal control in the degradation of the neurotransmitter acetylcholine (4Taylor P. Camp S. Radić Z. Acetylcholinesterase.in: Squire L.R. Encyclopedia of Neuroscience. 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P. at the acetylcholinesterase PubMed Scopus Google Scholar, The structural and of human PubMed Scopus Google Scholar, of of human acetylcholinesterase by and by 2018; PubMed Scopus Google Scholar). subunits of AChE P. Y. Camp S. Rachinsky T.L. Ekström T. Radić Z. and of of the acetylcholinesterase Biol. PubMed Scopus Google Scholar). The active serine in the catalytic in catalytic of the human AChE (hAChE) is at the of a Å active center The gorge is and and in the center of the to the acyl pocket Taylor P. Radić Z. A of human acetylcholinesterase for Biol. PubMed Scopus Google Scholar). The of homodimers, an of AChE quaternary is in X-ray of and Y. Taylor P. Radić Z. P. at the acetylcholinesterase PubMed Scopus Google Scholar, M. L. structure of acetylcholinesterase from Torpedo A 1991; PubMed Scopus Google Scholar). These show C-terminally from a dimerization by of disulfide-linked covalent homodimers AChE M. L. structure of acetylcholinesterase from Torpedo A 1991; PubMed Scopus Google and of reversibly associated AChE at the Y. Taylor P. Radić Z. P. at the acetylcholinesterase PubMed Scopus Google and quaternary homodimers also observed in X-ray of the structurally human (EC Y. P. structure of human and of its with and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and human E. P. E. of and the and Full Text Full Text PDF PubMed Scopus Google and P. G. Taylor P. Y. P. of the synaptic and its for and Full Text Full Text PDF PubMed Scopus Google and in the structure of human L. T. M. The structure of human PubMed Scopus Google Scholar). of hAChE and of demonstrated to with Taylor P. P. and of the the Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. The acetylcholinesterase region is for and of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of of dimers in to dissociation, the of and and localization of is the potential for biological associated with the dissociation of A structural on in the structure at the interface to dimer dissociation in solution was for the of hAChE The structural and of human PubMed Scopus Google Scholar). the present we small-angle X-ray scattering cryo-EM, and that a of wildtype hAChE the C-terminal forms reversible homodimers at micromolar concentrations. structurally to hAChE homodimers that linked covalently by C-terminal Y. Taylor P. Radić Z. P. at the acetylcholinesterase PubMed Scopus Google Scholar, M. L. structure of acetylcholinesterase from Torpedo A 1991; PubMed Scopus Google Scholar). of this C-terminally of hAChE to or the RP enantiomer of not or a variety of structurally diverse reversible ligands, promotes dissociation of the homodimers in solution as by Dissociation of reversible hAChE homodimers covalent binding of or the is likely the of structural in the acyl pocket of the AChE catalytic allosterically to the dimer interface 25 Å away. The structural effect be reversed by of the The biological of organophosphate structural in potential for of neurotransmitter in structural of the dissociation the of allosteric the active center of hAChE and the dimerization interface on the of the hAChE of this allosteric to the of AChE and its structural The and of to of catalytic of hAChE, at and and at the a molecular of to Y. Taylor P. Radić Z. P. at the acetylcholinesterase PubMed Scopus Google to a at micromolar this hAChE than hAChE may be of hAChE to the at larger volume to for and to for of this is consistent with the formation of reversible homodimers that to to concentrations. We with a of concentrations of recombinant monomeric hAChE in the of to to of hAChE by enzyme in where observed an dimer dissociation of to hAChE in the of the hAChE in wildtype or in in the of the hAChE double-mutant with The of effect to in the hAChE or the of to be consistent with the C-terminal a dimerization interface in homodimer formation homodimers observed in asymmetric subunits of the wildtype hAChE Z. Taylor P. and of of and Scopus Google Scholar). formation of a homodimer to the dimer and not to the dimer is consistent with of dimerization in this of hAChE that the dimer was the of the homodimers Taylor P. Radić Z. A of human acetylcholinesterase for Biol. 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Full Text Full Text PDF PubMed Scopus (13) Google at to sites in a of wildtype hAChE with profiles for of the reversible formation not on hAChE solution in of or of the or the in the or The reversible ligands, structurally binding sites the active center gorge of hAChE, for the acyl pocket These binding sites at the of the gorge and of the as and and of the where and The X-ray structure of the not it also to the it is to that binding of this at the of the gorge to the at its as in X-ray of of a of as as as the dimerization of hAChE not to be affected by with the gorge including the and of with of the hAChE and of the on the of the active center the and not to the dimerization interface at the of reversible the volume the acyl as by the of and The SAXS profiles of hAChE with a variety of to covalent at the active show that of the an increase in the of the and a reduction in the of the had no These in the by and the RP enantiomer of sarin a in the toward the covalent binding of the SAXS with that at homodimer and with homodimer for hAChE a dimer dissociation for of than the for hAChE The of and to a in the quaternary structure of hAChE be by and by where in the active of hAChE with in for covalent binding to on the the the acyl pocket of the hAChE The of and different The of is and to of its the acyl the of to its it to and to in a that its on the toward the acyl the and larger than the of and larger than the of the neurotransmitter of covalent with of in the and the that acyl pocket of the of the in the active is by is of than that of the acetylcholine Radić Z. 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The that in hAChE triggered by active by in the the of in covalent of AChE by occurs at the and not increase with of and acetylcholinesterase role of a binding PubMed Scopus Google Scholar, Z. Gibney G. S. MacPhee-Quigley K. Taylor P. of recombinant acetylcholinesterase in a of PubMed Scopus Google Scholar, of acetylcholinesterase by the organophosphate PubMed Scopus Google Scholar, T. Z. E. L. G. Z. Taylor P. acetylcholinesterase catalytic of that Biol. Chem. 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Anglister L. Fine localization of acetylcholinesterase in the synaptic cleft of the vertebrate neuromuscular junction.Front. Mol. Neurosci. 2018; 11: 123Crossref PubMed Scopus (13) Google Scholar). The consistent with dimerization through a of hAChE of in on hAChE at the and in a monomeric formation of a homodimer in the on the hAChE as the in this we a homodimer in to the for a of homodimers in the Taylor P. Radić Z. A of human acetylcholinesterase for Biol. PubMed Scopus Google Scholar, L. Taylor P. Radić Z. and of of human acetylcholinesterase in Chem. Biol. Chem. Full Text Full Text PDF PubMed Scopus (13) Google Scholar, T. Taylor P. Radić Z. of a in X-ray of native and human Biol. Chem. 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