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- PDB-7tsq: Structure of Enterobacter cloacae Cap2 bound to CdnD02 C-terminus... -

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Basic information

Entry
Database: PDB / ID: 7tsq
TitleStructure of Enterobacter cloacae Cap2 bound to CdnD02 C-terminus, AMP state
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 MONOPHOSPHATE / Cyclic AMP-AMP-GMP synthase
Similarity search - Component
Biological speciesEnterobacter cloacae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.11 Å
AuthorsYe, Q. / Gu, Y. / 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 31, 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
Revision 1.4Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.5Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)56,1737
Polymers55,4554
Non-polymers7193
Water3,927218
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7000 Å2
ΔGint-37 kcal/mol
Surface area16170 Å2
MethodPISA
Unit cell
Length a, b, c (Å)49.695, 76.354, 126.901
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Cap2


Mass: 26394.779 Da / Num. of mol.: 2 / Fragment: residues 363-600 / Mutation: C548A
Source method: isolated from a genetically manipulated source
Details: N-terminal SNA, C548A, C-terminal GSG / Source: (gene. exp.) Enterobacter cloacae (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein/peptide Cyclic AMP-AMP-GMP synthase / cGAS/DncV-like nucleotidyltransferase / CD-NTase038


Mass: 1332.526 Da / Num. of mol.: 2 / Fragment: residues 370-381
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
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: 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
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 218 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.17 Å3/Da / Density % sol: 43.34 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop
Details: 0.1 M Tris-HCl pH 8.5, 0.2 M MgCl2, and 30% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 3, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.11→126.9 Å / Num. obs: 28304 / % possible obs: 99.6 % / Redundancy: 6.5 % / Biso Wilson estimate: 35.74 Å2 / CC1/2: 0.993 / Rmerge(I) obs: 0.146 / Rpim(I) all: 0.062 / Net I/σ(I): 15.3
Reflection shellResolution: 2.11→2.18 Å / Rmerge(I) obs: 0.86 / Mean I/σ(I) obs: 2.5 / Num. unique obs: 2237 / CC1/2: 0.632 / Rpim(I) all: 0.384

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7TO3

7to3


Resolution: 2.11→65.42 Å / SU ML: 0.2059 / Cross valid method: FREE R-VALUE / σ(F): 0.31 / Phase error: 21.3722
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2293 1381 4.89 %
Rwork0.2022 26860 -
obs0.2035 28241 99.53 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 47.26 Å2
Refinement stepCycle: LAST / Resolution: 2.11→65.42 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3293 0 47 218 3558
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00813407
X-RAY DIFFRACTIONf_angle_d0.87844617
X-RAY DIFFRACTIONf_chiral_restr0.0531520
X-RAY DIFFRACTIONf_plane_restr0.007589
X-RAY DIFFRACTIONf_dihedral_angle_d11.80811214
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.11-2.190.24771400.25752558X-RAY DIFFRACTION97.4
2.19-2.280.25951390.24392669X-RAY DIFFRACTION99.96
2.28-2.380.2611170.22292662X-RAY DIFFRACTION99.96
2.38-2.510.21771290.20862675X-RAY DIFFRACTION99.89
2.51-2.660.25181520.22312641X-RAY DIFFRACTION99.68
2.66-2.870.24181260.24322657X-RAY DIFFRACTION99.46
2.87-3.160.28421570.22332685X-RAY DIFFRACTION99.65
3.16-3.620.20441360.20062699X-RAY DIFFRACTION99.89
3.62-4.560.19151360.16052756X-RAY DIFFRACTION99.83
4.56-65.420.23311490.19112858X-RAY DIFFRACTION99.6
Refinement TLS params.Method: refined / Origin x: -12.3714623366 Å / Origin y: -6.73785184655 Å / Origin z: 12.8618877577 Å
111213212223313233
T0.17947388677 Å2-0.0189490879686 Å20.0194774688761 Å2-0.214747820884 Å2-0.0285714392405 Å2--0.225300094274 Å2
L1.32885387849 °2-0.137723184716 °20.306380959974 °2-1.63568356188 °2-0.164098588477 °2--1.16191083133 °2
S8.14557548183E-5 Å °-0.139017585906 Å °0.259587703231 Å °0.120886436884 Å °-0.0164366123403 Å °0.113075484281 Å °-0.113726789895 Å °0.00552098250373 Å °-0.000191305400153 Å °
Refinement TLS groupSelection details: chain A or chain B or chain C or chain D

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