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- PDB-6n2m: NMR solution structure of the homodimeric, autoinhibited state of... -

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Basic information

Entry
Database: PDB / ID: 6n2m
TitleNMR solution structure of the homodimeric, autoinhibited state of the CARD9 CARD and first coiled-coil
ComponentsCaspase recruitment domain-containing protein 9
KeywordsSIGNALING PROTEIN / innate immunity / coiled-coil / autoinhibition
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 / positive regulation of innate immune response / positive regulation of T-helper 17 type immune response / neutrophil mediated immunity ...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 / positive regulation of innate immune response / positive regulation of T-helper 17 type immune response / neutrophil mediated immunity / positive regulation of cytokine production involved in inflammatory 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 / stress-activated MAPK cascade / 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 / regulation of apoptotic process / positive regulation of canonical NF-kappaB signal transduction / defense response to virus / 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 / cytoplasm / cytosol
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)
MethodSOLUTION NMR / simulated annealing
AuthorsHolliday, M.J. / Fairbrother, W.J. / Dueber, E.C.
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 17, 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
Revision 1.2Jun 14, 2023Group: Data collection / Database references / Other
Category: database_2 / pdbx_database_status / pdbx_nmr_spectrometer
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data / _pdbx_nmr_spectrometer.model
Revision 1.3May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Caspase recruitment domain-containing protein 9
B: Caspase recruitment domain-containing protein 9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,3644
Polymers32,2332
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2360 Å2
ΔGint-65 kcal/mol
Surface area15320 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

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


Mass: 16116.458 Da / Num. of mol.: 2 / Fragment: residues 2-142
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
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
113isotropic12D 1H-15N TROSY
122isotropic12D 1H-13C HSQC aliphatic
132isotropic12D 1H-13C TROSY aromatic
143isotropic13D HNCA
1121isotropic13D HNCA
153isotropic13D HNCB
1113isotropic13D CBCA(CO)NH
1133isotropic23D CBCA(CO)NH
1103isotropic13D HNCO
193isotropic13D HN(CA)CO
181isotropic13D 1H-15N NOESY
171isotropic13D (H)CC(CO)NH
1142isotropic13D (H)CCH-TOCSY
1152isotropic13D (H)CCH-COSY
1161isotropic13D 1H-13C NOESY aliphatic
1172isotropic13D 1H-13C NOESY aliphatic
1182isotropic13D 1H-13C NOESY aromatic
1191isotropic13D HNCO
1204isotropic13D 1H-13C interNOESY aliphatic
2215anisotropic12D IPAP 1H-15N TROSY

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution11 mM [U-13C; U-15N] CARD9_2-142, 1 mM Zinc, 95% H2O/5% D2O13C15N CARD9 2-142 in H2O13C15N_H2O_Sample95% H2O/5% D2O
solution21 mM [U-13C; U-15N] CARD9_2-142, 1 mM Zinc, 100% D2O13C15N CARD9 2-142 in D2O13C15N_D2O_Sample100% D2O
solution31 mM [U-13C; U-15N; U-2H] CARD9_2-142, 1 mM Zinc, 95% H2O/5% D2O2H13C15N CARD9 2-142 in H2O2H13C15N_H2O_Sample95% H2O/5% D2O
solution41 mM [U-13C; U-15N] CARD9_2-142, unlabeled 1 mM CARD9_2-142, 2 mM Zinc, 95% H2O/5% D2O50:50 12C14N:13C15N CARD9 2-142 in H2O50:50_13C15N_Sample95% H2O/5% D2O
filamentous virus51 mM [U-13C; U-15N] CARD9_2-142, 1 mM Zinc, 14 mg/mL filamentous Pf1 bacteriophage, 95% H2O/5% D2O13C15N_RDC_Sample95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1 mMCARD9_2-142[U-13C; U-15N]1
1 mMZincnatural abundance1
1 mMCARD9_2-142[U-13C; U-15N]2
1 mMZincnatural abundance2
1 mMCARD9_2-142[U-13C; U-15N; U-2H]3
1 mMZincnatural abundance3
1 mMCARD9_2-142[U-13C; U-15N]4
1 mMunlabeled CARD9_2-142natural abundance4
2 mMZincnatural abundance4
1 mMCARD9_2-142[U-13C; U-15N]5
1 mMZincnatural abundance5
14 mg/mLfilamentous Pf1 bacteriophagenatural abundance5
Sample conditions
Conditions-IDDetailsIonic strengthLabelpHPressure (kPa)Temperature (K)
150 mM HEPES, 300 mM NaCl, 0.5 mM TCEP, pH 7.0300 mMCondition_17.0 1 atm310 K
250 mM HEPES, 900 mM NaCl, 0.5 mM TCEP, pH 7.0900 mMCondition_27.0 1 atm310 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCEBrukerAVANCE8001
Bruker AVANCEBrukerAVANCE6002

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Processing

NMR software
NameVersionDeveloperClassification
CNS1.2Brunger A. T. et.al.refinement
CYANA3.97Guntert, Mumenthaler and Wuthrichrefinement
Analysis2.42CCPNchemical shift assignment
Analysis2.42CCPNpeak picking
CYANA3.97Guntert, Mumenthaler and Wuthrichstructure calculation
Refinement
MethodSoftware ordinal
simulated annealing2
simulated annealing1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 20

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