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- PDB-6x2j: Structure of human TRPA1 in complex with agonist GNE551 -

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Basic information

Entry
Database: PDB / ID: 6x2j
TitleStructure of human TRPA1 in complex with agonist GNE551
ComponentsTransient receptor potential cation channel subfamily A member 1
KeywordsMEMBRANE PROTEIN/Agonist / TRPA1 / channel / agonist / MEMBRANE PROTEIN / MEMBRANE PROTEIN-Agonist complex
Function / homology
Function and homology information


temperature-gated cation channel activity / thermoception / detection of chemical stimulus involved in sensory perception of pain / stereocilium bundle / channel activity / calcium-release channel activity / response to pain / response to organic substance / detection of mechanical stimulus involved in sensory perception of pain / ion transport ...temperature-gated cation channel activity / thermoception / detection of chemical stimulus involved in sensory perception of pain / stereocilium bundle / channel activity / calcium-release channel activity / response to pain / response to organic substance / detection of mechanical stimulus involved in sensory perception of pain / ion transport / calcium ion transmembrane transport / sensory perception of pain / response to cold / response to organic cyclic compound / response to hydrogen peroxide / protein homotetramerization / cell surface receptor signaling pathway / response to drug / integral component of plasma membrane / identical protein binding / plasma membrane
Ankyrin repeat-containing domain / Ankyrin repeat / Ion transport domain / Ankyrin repeat-containing domain superfamily
Transient receptor potential cation channel subfamily A member 1
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsRohou, A. / Rouge, L. / Chen, H.
CitationJournal: Neuron / Year: 2020
Title: A Non-covalent Ligand Reveals Biased Agonism of the TRPA1 Ion Channel.
Authors: Chang Liu / Rebecca Reese / Simon Vu / Lionel Rougé / Shannon D Shields / Satoko Kakiuchi-Kiyota / Huifen Chen / Kevin Johnson / Yu Patrick Shi / Tania Chernov-Rogan / Demi Maria Zabala ...Authors: Chang Liu / Rebecca Reese / Simon Vu / Lionel Rougé / Shannon D Shields / Satoko Kakiuchi-Kiyota / Huifen Chen / Kevin Johnson / Yu Patrick Shi / Tania Chernov-Rogan / Demi Maria Zabala Greiner / Pawan Bir Kohli / David Hackos / Bobby Brillantes / Christine Tam / Tianbo Li / Jianyong Wang / Brian Safina / Steve Magnuson / Matthew Volgraf / Jian Payandeh / Jie Zheng / Alexis Rohou / Jun Chen /
Abstract: The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent ...The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 20, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily A member 1
B: Transient receptor potential cation channel subfamily A member 1
C: Transient receptor potential cation channel subfamily A member 1
D: Transient receptor potential cation channel subfamily A member 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)292,2908
Polymers290,4894
Non-polymers1,8014
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Transient receptor potential cation channel subfamily A member 1 / Ankyrin-like with transmembrane domains protein 1 / Transformation-sensitive protein p120 / Wasabi receptor


Mass: 72622.125 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRPA1, ANKTM1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O75762
#2: Chemical
ChemComp-ULJ / 5-amino-1-[(4-bromo-2-fluorophenyl)methyl]-N-(2,5-dimethoxyphenyl)-1H-1,2,3-triazole-4-carboxamide


Mass: 450.262 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C18H17BrFN5O3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: TRPA1 bound by agonist GNE551 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8.2
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris1
2150 mMNaClSodium chlorideNaClSodium chloride1
30.5 mMTCEP1
SpecimenConc.: 0.33 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Solarus plasma cleaner / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Triple blot. Put blot in vitroblot Apply 3.5ul to grid, wait 30sec, blot manually Apply 3.5ul to grid, wait 30sec, blot manually, apply final 3.5ul, final blot by vitrobot (3.5s)

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.6 sec. / Electron dose: 41.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11063
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameCategoryDetails
2SerialEMimage acquisition
4cisTEMCTF correction
7Cootmodel fitting
8ISOLDEmodel fitting
10cisTEMinitial Euler assignment
11cisTEMfinal Euler assignment
12cisTEMclassification3D reconstruction
13PHENIXclassificationDensity modification
14cisTEM3D reconstruction
15PHENIXmodel refinement
16Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 505112
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58837 / Algorithm: FOURIER SPACE
Details: Data at spatial frequencies higher than 1/4.0 A-1 were not used during any part of the refinement. The final map was density-modified using Phenix.
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6PQQ

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