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- PDB-6puu: Human TRPM2 bound to 8-Br-cADPR and calcium -

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Basic information

Entry
Database: PDB / ID: 6puu
TitleHuman TRPM2 bound to 8-Br-cADPR and calcium
ComponentsTransient receptor potential cation channel subfamily M member 2
KeywordsTRANSPORT PROTEIN / TRPM2 channel / 8-Br-cADPR / calcium
Function / homology
Function and homology information


response to purine-containing compound / cellular response to purine-containing compound / mono-ADP-D-ribose binding / zinc ion transmembrane transport / cellular response to temperature stimulus / ligand-gated calcium channel activity / cation transmembrane transport / dendritic cell differentiation / metal ion transport / regulation of filopodium assembly ...response to purine-containing compound / cellular response to purine-containing compound / mono-ADP-D-ribose binding / zinc ion transmembrane transport / cellular response to temperature stimulus / ligand-gated calcium channel activity / cation transmembrane transport / dendritic cell differentiation / metal ion transport / regulation of filopodium assembly / temperature homeostasis / sodium channel activity / calcium ion transmembrane import into cytosol / TRP channels / calcium ion import across plasma membrane / regulation of actin cytoskeleton reorganization / dendritic cell chemotaxis / calcium-release channel activity / ficolin-1-rich granule membrane / tertiary granule membrane / release of sequestered calcium ion into cytosol / calcium-mediated signaling using intracellular calcium source / cation channel activity / specific granule membrane / cellular response to calcium ion / calcium channel activity / calcium ion transmembrane transport / cell projection / calcium ion transport / cellular response to hydrogen peroxide / lysosomal membrane / cytoplasmic vesicle membrane / perikaryon / protein homotetramerization / lysosome / Neutrophil degranulation / neutrophil degranulation / calcium ion binding / integral component of plasma membrane / plasma membrane
Similarity search - Function
Transient receptor potential cation channel subfamily M member 2 / SLOG in TRPM / TRPM, SLOG domain / NUDIX hydrolase domain / Nudix hydrolase domain profile. / NUDIX hydrolase-like domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Chem-CV1 / Transient receptor potential cation channel subfamily M member 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsDu, J. / Lu, W. / Huang, Y.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS111031 United States
CitationJournal: Elife / Year: 2019
Title: Ligand recognition and gating mechanism through three ligand-binding sites of human TRPM2 channel.
Authors: Yihe Huang / Becca Roth / Wei Lü / Juan Du /
Abstract: TRPM2 is critically involved in diverse physiological processes including core temperature sensing, apoptosis, and immune response. TRPM2's activation by Ca and ADP ribose (ADPR), an NAD-metabolite ...TRPM2 is critically involved in diverse physiological processes including core temperature sensing, apoptosis, and immune response. TRPM2's activation by Ca and ADP ribose (ADPR), an NAD-metabolite produced under oxidative stress and neurodegenerative conditions, suggests a role in neurological disorders. We provide a central concept between triple-site ligand binding and the channel gating of human TRPM2. We show consecutive structural rearrangements and channel activation of TRPM2 induced by binding of ADPR in two indispensable locations, and the binding of Ca in the transmembrane domain. The 8-Br-cADPR-an antagonist of cADPR-binds only to the MHR1/2 domain and inhibits TRPM2 by stabilizing the channel in an apo-like conformation. We conclude that MHR1/2 acts as a orthostatic ligand-binding site for TRPM2. The NUDT9-H domain binds to a second ADPR to assist channel activation in vertebrates, but not necessary in invertebrates. Our work provides insights into the gating mechanism of human TRPM2 and its pharmacology.
History
DepositionJul 18, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Assembly

Deposited unit
A: Transient receptor potential cation channel subfamily M member 2
B: Transient receptor potential cation channel subfamily M member 2
C: Transient receptor potential cation channel subfamily M member 2
D: Transient receptor potential cation channel subfamily M member 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)691,76512
Polymers689,1204
Non-polymers2,6458
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18840 Å2
ΔGint-226 kcal/mol
Surface area249500 Å2

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Components

#1: Protein
Transient receptor potential cation channel subfamily M member 2 / TRPM2 / Estrogen-responsive element-associated gene 1 protein / Long transient receptor potential ...TRPM2 / Estrogen-responsive element-associated gene 1 protein / Long transient receptor potential channel 2 / LTrpC2 / Transient receptor potential channel 7 / TrpC7 / Transient receptor potential melastatin 2


Mass: 172280.062 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TRPM2, EREG1, KNP3, LTRPC2, TRPC7 / Production host: Homo sapiens (human) / References: UniProt: O94759
#2: Chemical
ChemComp-CV1 / (2R,3R,4S,5R,13R,14S,15R,16R)-24-amino-18-bromo-3,4,14,15-tetrahydroxy-7,9,11,25,26-pentaoxa-17,19,22-triaza-1-azonia-8,10-diphosphapentacyclo[18.3.1.1^2,5^.1^13,16^.0^17,21^]hexacosa-1(24),18,20,22-tetraene-8,10-diolate 8,10-dioxide / 8-BROMO-CYCLIC-ADP-RIBOSE


Mass: 621.204 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C15H21BrN5O13P2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CA / CALCIUM ION / Calcium


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca / 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: human TRPM2 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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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 defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8 sec. / Electron dose: 6.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4GctfCTF correction
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 102259 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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