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- PDB-8qvn: Cryo-EM structure of Cx26 from Gallus Gallus in bicarbonate buffer -

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Basic information

Entry
Database: PDB / ID: 8qvn
TitleCryo-EM structure of Cx26 from Gallus Gallus in bicarbonate buffer
ComponentsGap junction protein
KeywordsMEMBRANE PROTEIN / Gap junction Large Pore Channel Carbon dioxide sensitive
Function / homology
Function and homology information


Gap junction assembly / gap junction-mediated intercellular transport / connexin complex / gap junction channel activity / sensory perception of sound / cell-cell signaling
Similarity search - Function
Connexin / Connexin, N-terminal / Connexin, conserved site / Gap junction protein, cysteine-rich domain / Connexin, N-terminal domain superfamily / Connexin / Connexins signature 1. / Connexins signature 2. / Connexin homologues / Gap junction channel protein cysteine-rich domain
Similarity search - Domain/homology
PHOSPHATIDYLETHANOLAMINE / Gap junction protein
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.07 Å
AuthorsBrotherton, D.H. / Cameron, A.D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/P010393/1 United Kingdom
Leverhulme TrustRPG-2015-090 United Kingdom
CitationJournal: J Physiol / Year: 2025
Title: Multiple carbamylation events are required for differential modulation of Cx26 hemichannels and gap junctions by CO.
Authors: Sarbjit Nijjar / Deborah Brotherton / Jack Butler / Valentin-Mihai Dospinescu / Harry G Gannon / Victoria Linthwaite / Martin Cann / Alexander Cameron / Nicholas Dale /
Abstract: CO directly modifies the gating of connexin26 (Cx26) gap junction channels and hemichannels. This gating depends upon Lys125, and the proposed mechanism involves carbamylation of Lys125 to allow ...CO directly modifies the gating of connexin26 (Cx26) gap junction channels and hemichannels. This gating depends upon Lys125, and the proposed mechanism involves carbamylation of Lys125 to allow formation of a salt bridge with Arg104 on the neighbouring subunit. We demonstrate via carbamate trapping and tandem mass spectrometry that five Lys residues within the cytoplasmic loop, including Lys125, are indeed carbamylated by CO. The cytoplasmic loop appears to provide a chemical microenvironment that facilitates carbamylation. Systematic mutation of these Lys residues to Arg shows that only carbamylation of Lys125 is essential for hemichannel opening. By contrast, carbamylation of Lys108 and Lys125 is essential for gap junction closure to CO. Chicken (Gallus gallus) Cx26 gap junction channels lack Lys108 and do not close to CO, as shown by both a dye transfer assay and a high-resolution cryogenic electron microscopy structure. The mutation Lys108Arg prevents CO-dependent gap junction channel closure in human Cx26. Our findings directly demonstrate carbamylation in connexins, provide further insight into the differential action of CO on Cx26 hemichannels and gap junction channels, and increase support for the role of the N-terminus in gating the Cx26 channel. KEY POINTS: Direct evidence of carbamylation of multiple lysine residues in the cytoplasmic loop of Cx26. Concentration-dependent carbamylation at lysines 108, 122 and 125. Only carbamylation of lysine 125 is essential for hemichannel opening to CO. Carbamylation of lysine 108 along with lysine 125 is essential for CO-dependent gap junction channel closure.
History
DepositionOct 18, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Gap junction protein
B: Gap junction protein
C: Gap junction protein
D: Gap junction protein
E: Gap junction protein
F: Gap junction protein
G: Gap junction protein
H: Gap junction protein
I: Gap junction protein
J: Gap junction protein
K: Gap junction protein
L: Gap junction protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)345,04160
Polymers315,16912
Non-polymers29,87248
Water8,395466
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Gap junction protein


Mass: 26264.119 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: GJB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: E1C2J9
#2: Sugar...
ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM
#3: Chemical...
ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 466 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
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: Connexin 26 (Gallus Gallus) in 90mmHg PCO2, pH7.4 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Details: The buffer, except DDM and DTT, was prepared fresh from 10x stock on day of used. The basal buffer was filtered and degassed and DDM and DTT added. The buffer was pH corrected at point of ...Details: The buffer, except DDM and DTT, was prepared fresh from 10x stock on day of used. The basal buffer was filtered and degassed and DDM and DTT added. The buffer was pH corrected at point of use to pH 7.4 using 15% CO2 in 85% N2.
Buffer component
IDConc.NameFormulaBuffer-ID
170 mMsodium chlorideNaCl1
25 %glycerolC3H8O31
31 mMDTTC4H10O2S21
40.03 %DDMC2H46O111
580 mMsodium hydorgen carbonateNaH2P041
63 mMpotassium chlorideKCl1
71 mMmagnesium sulphateMgSO41
84 mMmagnesium chlorideMgCl21
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE-PROPANE
Details: 3 microlitres protein applied to grid, blot time 7 seconds, in 15% CO2/85%N2 atmosphere

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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 FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 41 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 8713
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectionbeta
4CTFFIND4CTF correction
7Coot0.9.8.4model fitting
9PHENIX1.20.1model refinement
13RELION3.13D reconstructionbeta
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2041681
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 2.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161947 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 7QEQ

7qeq


Accession code: 7QEQ / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 84.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002620532
ELECTRON MICROSCOPYf_angle_d0.48227696
ELECTRON MICROSCOPYf_chiral_restr0.03843060
ELECTRON MICROSCOPYf_plane_restr0.00243300
ELECTRON MICROSCOPYf_dihedral_angle_d7.7163120

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