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- PDB-9bz8: Pannexin 1 containing C-terminal activating domain -

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Basic information

Entry
Database: PDB / ID: 9bz8
TitlePannexin 1 containing C-terminal activating domain
ComponentsPannexin
KeywordsMEMBRANE PROTEIN / ATP release channel / large-pore / nanodisc / C-terminal activating domain
Function / homology
Function and homology information


positive regulation of interleukin-1 production / gap junction / monoatomic cation transport / channel activity / monoatomic ion transmembrane transport / endoplasmic reticulum membrane / plasma membrane
Similarity search - Function
Pannexin / Innexin / Innexin / Pannexin family profile.
Similarity search - Domain/homology
Biological speciesXenopus tropicalis (tropical clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsEhrlich, J.J. / Kawate, T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM114379 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: The C-terminal activating domain promotes pannexin 1 channel opening.
Authors: Erik Henze / Jacqueline J Ehrlich / Janice L Robertson / Eric Gelsleichter / Toshimitsu Kawate /
Abstract: Pannexin 1 (Panx1) constitutes a large pore channel responsible for the release of adenosine triphosphate (ATP) from apoptotic cells. Strong evidence indicates that caspase-mediated cleavage of the C- ...Pannexin 1 (Panx1) constitutes a large pore channel responsible for the release of adenosine triphosphate (ATP) from apoptotic cells. Strong evidence indicates that caspase-mediated cleavage of the C-terminus promotes the opening of the Panx1 channel by unplugging the pore. However, this simple pore-plugging mechanism alone cannot account for the observation that a Panx1 construct ending before the caspase cleavage site remains closed. Here, we show that a helical region located immediately before the caspase cleavage site, referred to as the "C-terminal activating domain (CAD)", plays a pivotal role in facilitating Panx1 activation. Electrophysiology and mutagenesis studies uncovered that two conserved leucine residues within the CAD play a pivotal role. Cryoelectron microscopy (Cryo-EM) analysis of the construct ending before reaching the CAD demonstrated that the N terminus extends into an intracellular pocket. In contrast, the construct including the CAD revealed that this domain occupies the intracellular pocket, causing the N terminus to flip upward within the pore. Analysis of electrostatic free energy landscape in the closed conformation indicated that the intracellular side of the ion permeation pore may be occupied by anions like ATP, creating an electrostatic barrier for anions attempting to permeate the pore. When the N terminus flips up, it diminishes the positively charged surface, thereby reducing the drive to accumulate anions inside the pore. This dynamic change in the electrostatic landscape likely contributes to the selection of permeant ions. Collectively, these experiments put forth a mechanism in which C-terminal cleavage liberates the CAD, causing the repositioning of the N terminus to promote Panx1 channel opening.
History
DepositionMay 24, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pannexin
B: Pannexin
C: Pannexin
D: Pannexin
E: Pannexin
F: Pannexin
G: Pannexin


Theoretical massNumber of molelcules
Total (without water)299,6907
Polymers299,6907
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Pannexin


Mass: 42812.848 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus tropicalis (tropical clawed frog)
Gene: panx1, PANX / Plasmid: pFBNT / Details (production host): Internal strepII tag / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A803JW22
Has protein modificationY

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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: frog pannexin 1 / Type: COMPLEX
Details: Engineered frog pannexin 1 containing c-terminal activating domain in lipid nanodiscs
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 43.5 kDa/nm / Experimental value: NO
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: BTI-Tn-5B1-4
Buffer solutionpH: 7.4
Details: Sample was subject to size exclusion chromatography with HEPES/NaCl buffer.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: frPanx1 in lipid nanodiscs containing POPC, POPG, POPE and cholesterol. Sample eluted in a single peak from size-exclusion and was pooled and concentrated.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288.15 K
Details: Grid was blotted for 4 seconds with a force of 7 before plunging into liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 20000 nm / Nominal defocus min: 8000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.27 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1
Details: 50-frame movies were collected in counted super resolution mode.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 15 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4particle selectiontemplate picker with templates generated from previous Pannexin maps used to pick particles
2EPUimage acquisition
4cryoSPARC4CTF correction
5RELION4CTF correction
8Coot0.8.9.1model fitting
10RELION4initial Euler assignment
11RELION4final Euler assignment
12RELION4classification
13RELION4.0-beta3D reconstruction
14PHENIX1.21.1-5286model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10234069
Details: Particles picked with template picker and inspect particle picks was deployed to remove non-protein picks
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31614 / Algorithm: FOURIER SPACE
Details: Reconstructed in RELION using 3D classification and autorefinement
Num. of class averages: 8 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6VD7

6vd7


Accession code: 6VD7 / Details: Complete assembly of PDB entry 6VD7 / Source name: PDB / Type: experimental model

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