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- PDB-6n2p: Helical assembly of the CARD9 CARD -

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Basic information

Entry
Database: PDB / ID: 6n2p
TitleHelical assembly of the CARD9 CARD
ComponentsCaspase recruitment domain-containing protein 9
KeywordsSIGNALING PROTEIN / CARD / filament / helical assembly / death domain / innate immunity
Function / homology
Function and homology information


regulation of interleukin-2 production / host-mediated regulation of intestinal microbiota composition / CBM complex / antifungal innate immune response / response to peptidoglycan / positive regulation of stress-activated MAPK cascade / CARD domain binding / neutrophil mediated immunity / positive regulation of cytokine production involved in inflammatory response / positive regulation of innate immune response ...regulation of interleukin-2 production / host-mediated regulation of intestinal microbiota composition / CBM complex / antifungal innate immune response / response to peptidoglycan / positive regulation of stress-activated MAPK cascade / CARD domain binding / neutrophil mediated immunity / positive regulation of cytokine production involved in inflammatory response / positive regulation of innate immune response / positive regulation of T-helper 17 type immune response / positive regulation of granulocyte macrophage colony-stimulating factor production / positive regulation of macrophage cytokine production / response to aldosterone / response to exogenous dsRNA / positive regulation of interleukin-17 production / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / response to muramyl dipeptide / immunoglobulin mediated immune response / positive regulation of chemokine production / JNK cascade / positive regulation of cytokine production / positive regulation of JNK cascade / NOD1/2 Signaling Pathway / protein homooligomerization / CLEC7A (Dectin-1) signaling / positive regulation of interleukin-6 production / : / positive regulation of tumor necrosis factor production / positive regulation of NF-kappaB transcription factor activity / defense response to virus / regulation of apoptotic process / positive regulation of canonical NF-kappaB signal transduction / positive regulation of ERK1 and ERK2 cascade / defense response to Gram-positive bacterium / response to xenobiotic stimulus / protein homodimerization activity / protein-containing complex / identical protein binding / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
CARD9, CARD domain / CARD domain / CARD caspase recruitment domain profile. / Caspase recruitment domain / Death-like domain superfamily
Similarity search - Domain/homology
Caspase recruitment domain-containing protein 9
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4 Å
AuthorsHolliday, M.J. / Rohou, A. / Arthur, C.P. / Dueber, E.C. / Fairbrother, W.J.
CitationJournal: Nat Commun / Year: 2019
Title: Structures of autoinhibited and polymerized forms of CARD9 reveal mechanisms of CARD9 and CARD11 activation.
Authors: Michael J Holliday / Axel Witt / Alejandro Rodríguez Gama / Benjamin T Walters / Christopher P Arthur / Randal Halfmann / Alexis Rohou / Erin C Dueber / Wayne J Fairbrother /
Abstract: CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this ...CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.
History
DepositionNov 13, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 3, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Caspase recruitment domain-containing protein 9
B: Caspase recruitment domain-containing protein 9
C: Caspase recruitment domain-containing protein 9
D: Caspase recruitment domain-containing protein 9
E: Caspase recruitment domain-containing protein 9
F: Caspase recruitment domain-containing protein 9
G: Caspase recruitment domain-containing protein 9
H: Caspase recruitment domain-containing protein 9
I: Caspase recruitment domain-containing protein 9
J: Caspase recruitment domain-containing protein 9


Theoretical massNumber of molelcules
Total (without water)173,51910
Polymers173,51910
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12790 Å2
ΔGint-5 kcal/mol
Surface area44960 Å2
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 10 / Rise per n subunits: 5.11 Å / Rotation per n subunits: -101.6 °)

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Components

#1: Protein
Caspase recruitment domain-containing protein 9 / hCARD9


Mass: 17351.873 Da / Num. of mol.: 10 / Mutation: I107E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CARD9 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q9H257

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Helical assembly of the CARD9 CARD. / Type: COMPLEX
Details: Formed from CARD9 2-152 dimer with an I107E mutation, purified with 1:1 Zn. 1 mM EDTA was added to 0.5 mM protein, followed by 10 minute incubation at 25C.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 34.0 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMSodium ChlorideNaClSodium chloride1
30.5 mMTCEPC9H15O6P1
40.5 mMZincZn1
51 mMEDTAEthylenediaminetetraacetic acidC10H16N2O81
SpecimenConc.: 8.67 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Formed from CARD9 2-152 dimer with an I107E mutation, purified with 1:1 Zn. 1 mM EDTA was added to 0.5 mM protein, followed by 10 minute incubation at 25C. Sample was subsequently diluted 1: ...Details: Formed from CARD9 2-152 dimer with an I107E mutation, purified with 1:1 Zn. 1 mM EDTA was added to 0.5 mM protein, followed by 10 minute incubation at 25C. Sample was subsequently diluted 1:5 before adding to grid.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Sample was incubated on the grid for 1 minute, subsequently washed/blotted 6 times in buffer, followed by addition of 3.5 ul buffer, which was blotted by vitrobot for 5 seconds prior to plunging.

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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: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1250 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 10 sec. / Electron dose: 50.9 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4403 / Details: Collected in movie-mode at 4 frames per second
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN2particle selectionFilaments were manually selected using e2helixboxer.py.
2RELION2.1particle selectionSegments were extracted from filaments using RELION.
3SerialEMimage acquisition
5CTFFINDCTF correctionCTFfind was used, implemented in cisTEM 1.0.0.
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
11Cootmodel refinement
12RELION2.1initial Euler assignment
13FREALIGNinitial Euler assignmentFrealign, as implemented by cisTEM 1.0.0.
14FREALIGN9.11final Euler assignment
15FREALIGN9.11classification
16FREALIGN9.113D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -101.6 ° / Axial rise/subunit: 5.11 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 141592
Details: Filaments were manually selected using e2helixboxer.py. Segments were extracted from filaments using RELION, using a 30 A shift between particles.
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31908
Details: A 5.0 A high-resolution limit was used for particle alignment in frealign.
Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: "Fit in map" in UCSF Chimera was used to fit the previously determined, lowest energy NMR solution structure (PDB ID 6E26) into the sharpened density and to generate 9 symmetry-mates ...Details: "Fit in map" in UCSF Chimera was used to fit the previously determined, lowest energy NMR solution structure (PDB ID 6E26) into the sharpened density and to generate 9 symmetry-mates according to the determined helical parameters. The map was then iteratively refined in Phenix and Coot, maintaining strict non-crystallographic symmetry among the CARDs.
Atomic model buildingPDB-ID: 6.0E+26 / Pdb chain-ID: A / Accession code: 6.0E+26 / Pdb chain residue range: 8-95 / Source name: PDB / Type: experimental model

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