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- PDB-7to3: Structure of Enterobacter cloacae Cap2-CdnD02 2:2 complex -

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Basic information

Entry
Database: PDB / ID: 7to3
TitleStructure of Enterobacter cloacae Cap2-CdnD02 2:2 complex
Components
  • Cap2
  • Cyclic AMP-AMP-GMP synthase
KeywordsTRANSFERASE / CBASS / ubiquitin E1/E2 / bacterial anti-phage defense / cGAS
Function / homology
Function and homology information


nucleotide metabolic process / nucleotidyltransferase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / defense response to virus / GTP binding / ATP binding / metal ion binding
Similarity search - Function
Second Messenger Oligonucleotide or Dinucleotide Synthetase domain / Nucleotidyltransferase superfamily
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE MONOPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Cyclic AMP-AMP-GMP synthase
Similarity search - Component
Biological speciesEnterobacter cloacae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsGu, Y. / Ye, Q. / Ledvina, H.E. / Quan, Y. / Lau, R.K. / Zhou, H. / Whiteley, A.T. / Corbett, K.D.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM104141 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21 AI148814 United States
CitationJournal: Nature / Year: 2023
Title: An E1-E2 fusion protein primes antiviral immune signalling in bacteria.
Authors: Hannah E Ledvina / Qiaozhen Ye / Yajie Gu / Ashley E Sullivan / Yun Quan / Rebecca K Lau / Huilin Zhou / Kevin D Corbett / Aaron T Whiteley /
Abstract: In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor ...In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor cyclic GMP-AMP synthase (cGAS) detects viral infection to produce the nucleotide second messenger cyclic GMP-AMP (cGAMP), which initiates stimulator of interferon genes (STING)-dependent antiviral signalling. Bacteria encode evolutionary predecessors of cGAS called cGAS/DncV-like nucleotidyltransferases (CD-NTases), which detect bacteriophage infection and produce diverse nucleotide second messengers. How bacterial CD-NTase activation is controlled remains unknown. Here we show that CD-NTase-associated protein 2 (Cap2) primes bacterial CD-NTases for activation through a ubiquitin transferase-like mechanism. A cryo-electron microscopy structure of the Cap2-CD-NTase complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling eukaryotic E1 and E2 proteins. The structure captures a reactive-intermediate state with the CD-NTase C terminus positioned in the Cap2 E1 active site and conjugated to AMP. Cap2 conjugates the CD-NTase C terminus to a target molecule that primes the CD-NTase for increased cGAMP production. We further demonstrate that a specific endopeptidase, Cap3, balances Cap2 activity by cleaving CD-NTase-target conjugates. Our data demonstrate that bacteria control immune signalling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of the E1 and E2 machinery across domains of life.
History
DepositionJan 22, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Feb 22, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Apr 26, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cap2
B: Cap2
C: Cyclic AMP-AMP-GMP synthase
D: Cyclic AMP-AMP-GMP synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)227,72414
Polymers225,0644
Non-polymers2,66010
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_1ens_2chain "D"
d_2ens_2chain "C"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLNTYRA1 - 568
d_21ens_1GLNTYRB1 - 568
d_11ens_2GLUALAI1 - 357
d_12ens_2AMPAMPJ
d_13ens_2MGMGK
d_14ens_2MGMGL
d_15ens_2ATPATPM
d_16ens_2ADPADPN
d_21ens_2GLUALAC1 - 357
d_22ens_2AMPAMPD
d_23ens_2MGMGE
d_24ens_2MGMGF
d_25ens_2ATPATPG
d_26ens_2ADPADPH

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(-0.999997334543, -0.0022230085571, -0.000623811283807), (0.00222404460418, -0.999996140552, -0.00166508461462), (-0.000620107378893, -0.00166646756053, 0.999998419175)370.151462605, 369.464749544, 0.473677726643
2given(-0.999996083941, 0.0027948629825, -0.000144374173926), (-0.00279452481652, -0.999993469427, -0.00229166645904), (-0.000150778124835, -0.00229125402753, 0.999997363707)369.146698448, 370.512295656, 0.403678746104

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Components

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Protein , 2 types, 4 molecules ABCD

#1: Protein Cap2


Mass: 67024.953 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacter cloacae (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein Cyclic AMP-AMP-GMP synthase / cGAS/DncV-like nucleotidyltransferase / CD-NTase038


Mass: 45506.828 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacter cloacae (bacteria) / Gene: cdnD02, P853_02262 / Production host: Escherichia coli (E. coli)
References: UniProt: P0DSP4, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases

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Non-polymers , 4 types, 10 molecules

#3: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

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: Quaternary complex of Cap2 and CdnD02 in a 2:2 stoichiometry, with CdnD02 substrate captured at the active site of Cap2.
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.2248 MDa / Experimental value: NO
Source (natural)Organism: Enterobacter cloacae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.5
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingAverage exposure time: 10 sec. / Electron dose: 64.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1cryoSPARC3.2.0particle selection
4cryoSPARC3.2.0CTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10cryoSPARC3.2.0initial Euler assignment
12cryoSPARC3.2.0classification
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138202 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 7LJL

7ljl


Pdb chain-ID: A
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 99.86 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001815150
ELECTRON MICROSCOPYf_angle_d0.51220654
ELECTRON MICROSCOPYf_chiral_restr0.03742250
ELECTRON MICROSCOPYf_plane_restr0.00422648
ELECTRON MICROSCOPYf_dihedral_angle_d6.06272026
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000707314898579
ens_2d_2DELECTRON MICROSCOPYNCS constraints0.000700096616647

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