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- EMDB-24404: Covalent inhibition of hAChE by organophosphates causes homodimer... -

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Basic information

Entry
Database: EMDB / ID: EMD-24404
TitleCovalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects
Map dataLow-resolution cryo-EM map of dimeric human acetylcholinesterase at 9 Angstrom.
Sample
  • Complex: Acetylcholinesterase
Function / homology
Function and homology information


negative regulation of synaptic transmission, cholinergic / Neurotransmitter clearance / acetylcholine catabolic process in synaptic cleft / cholinesterase activity / serine hydrolase activity / acetylcholine catabolic process / acetylcholine binding / amyloid precursor protein metabolic process / acetylcholinesterase / acetylcholine receptor signaling pathway ...negative regulation of synaptic transmission, cholinergic / Neurotransmitter clearance / acetylcholine catabolic process in synaptic cleft / cholinesterase activity / serine hydrolase activity / acetylcholine catabolic process / acetylcholine binding / amyloid precursor protein metabolic process / acetylcholinesterase / acetylcholine receptor signaling pathway / osteoblast development / acetylcholinesterase activity / choline metabolic process / Synthesis of PC / basement membrane / regulation of receptor recycling / Synthesis, secretion, and deacylation of Ghrelin / side of membrane / synaptic cleft / laminin binding / synapse assembly / collagen binding / positive regulation of protein secretion / neuromuscular junction / receptor internalization / : / retina development in camera-type eye / nervous system development / positive regulation of cold-induced thermogenesis / amyloid-beta binding / hydrolase activity / cell adhesion / synapse / perinuclear region of cytoplasm / Golgi apparatus / cell surface / protein homodimerization activity / extracellular space / extracellular region / membrane / nucleus / plasma membrane
Similarity search - Function
Acetylcholinesterase, tetramerisation domain / Acetylcholinesterase tetramerisation domain / Cholinesterase / Carboxylesterase type B, conserved site / Carboxylesterases type-B signature 2. / Carboxylesterase type B, active site / Carboxylesterases type-B serine active site. / Carboxylesterase, type B / Carboxylesterase family / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Acetylcholinesterase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsBlumenthal DK / Cheng X / Fager M / Ho KY / Rohrer J / Gerlits O / Juneja P / Kovalevsky A / Radic Z
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R21 NS098998 United States
CitationJournal: J Biol Chem / Year: 2021
Title: Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects.
Authors: Donald K Blumenthal / Xiaolin Cheng / Mikolai Fajer / Kwok-Yiu Ho / Jacqueline Rohrer / Oksana Gerlits / Palmer Taylor / Puneet Juneja / Andrey Kovalevsky / Zoran Radić /
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- ...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 R 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 S-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.
History
DepositionJul 7, 2021-
Header (metadata) releaseJul 28, 2021-
Map releaseJul 28, 2021-
UpdateSep 1, 2021-
Current statusSep 1, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.281
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.281
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24404.png

Downloads & links

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Map

FileDownload / File: emd_24404.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLow-resolution cryo-EM map of dimeric human acetylcholinesterase at 9 Angstrom.
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.281 / Movie #1: 0.281
Minimum - Maximum-0.25179586 - 0.7246119
Average (Standard dev.)0.0029441882 (±0.044145096)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 270.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z270.400270.400270.400
α/β/γ90.00090.00090.000
start NX/NY/NZ127131139
NX/NY/NZ1079278
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.2520.7250.003

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Supplemental data

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Mask #1

Fileemd_24404_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Acetylcholinesterase

EntireName: Acetylcholinesterase
Components
  • Complex: Acetylcholinesterase

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Supramolecule #1: Acetylcholinesterase

SupramoleculeName: Acetylcholinesterase / type: complex / ID: 1 / Parent: 0
Details: Gnt1- HEK293 mammalian cell culture was used to express hAChE
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 Gnt1-

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 10 mM HEPES, pH 7.4, 10 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Details: Cp3-Plunger.
Details0.2

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 64.56 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: Ab Initio model using Cryosparc
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2744
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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