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- PDB-7mir: Cryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex -

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Basic information

Entry
Database: PDB / ID: 7mir
TitleCryo-EM structure of SidJ-SdeA-CaM reaction intermediate complex
Components
  • Calmodulin-2
  • Calmodulin-dependent glutamylase SidJ
  • Ubiquitinating/deubiquitinating enzyme SdeA
KeywordsTRANSFERASE / HYDROLASE/LIGASE / SidJ / SdeA / CaM / complex / Intermediate / Acyl / Adenylate / Legionella / Ubiquitination / HYDROLASE-LIGASE complex
Function / homology
Function and homology information


Ligases / NAD+-protein-arginine ADP-ribosyltransferase / negative regulation of calcium ion transmembrane transporter activity / deNEDDylase activity / NAD+-protein-arginine ADP-ribosyltransferase activity / protein deneddylation / Transferases; Acyltransferases; Aminoacyltransferases / host cell / K63-linked deubiquitinase activity / negative regulation of calcium ion export across plasma membrane ...Ligases / NAD+-protein-arginine ADP-ribosyltransferase / negative regulation of calcium ion transmembrane transporter activity / deNEDDylase activity / NAD+-protein-arginine ADP-ribosyltransferase activity / protein deneddylation / Transferases; Acyltransferases; Aminoacyltransferases / host cell / K63-linked deubiquitinase activity / negative regulation of calcium ion export across plasma membrane / positive regulation of ryanodine-sensitive calcium-release channel activity / regulation of cell communication by electrical coupling involved in cardiac conduction / negative regulation of peptidyl-threonine phosphorylation / protein deubiquitination / protein phosphatase activator activity / positive regulation of cyclic-nucleotide phosphodiesterase activity / positive regulation of phosphoprotein phosphatase activity / ligase activity / adenylate cyclase binding / catalytic complex / detection of calcium ion / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / voltage-gated potassium channel complex / cysteine-type peptidase activity / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / regulation of calcium-mediated signaling / positive regulation of protein dephosphorylation / titin binding / positive regulation of protein autophosphorylation / sperm midpiece / calcium channel complex / substantia nigra development / adenylate cyclase activator activity / regulation of heart rate / nucleotidyltransferase activity / protein serine/threonine kinase activator activity / sarcomere / positive regulation of peptidyl-threonine phosphorylation / regulation of cytokinesis / spindle microtubule / positive regulation of protein serine/threonine kinase activity / spindle pole / response to calcium ion / calcium-dependent protein binding / G2/M transition of mitotic cell cycle / myelin sheath / transferase activity / vesicle / transmembrane transporter binding / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / protein ubiquitination / G protein-coupled receptor signaling pathway / nucleotide binding / centrosome / calcium ion binding / protein kinase binding / protein-containing complex / proteolysis / extracellular region / membrane / metal ion binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
SidE, DUB domain / SidE, mono-ADP-ribosyltransferase domain / SidE mono-ADP-ribosyltransferase domain / SidE DUB domain / SidE, PDE domain / SidE phosphodiesterase (PDE) domain / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. ...SidE, DUB domain / SidE, mono-ADP-ribosyltransferase domain / SidE mono-ADP-ribosyltransferase domain / SidE DUB domain / SidE, PDE domain / SidE phosphodiesterase (PDE) domain / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
ADENOSINE MONOPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Calmodulin-2 / Ubiquitinating/deubiquitinating enzyme SdeA / Calmodulin-dependent glutamylase SidJ
Similarity search - Component
Biological speciesLegionella pneumophila (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsOsinski, A. / Black, M.H. / Pawlowski, K. / Chen, Z. / Li, Y. / Tagliabracci, V.S.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2GM137419 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)F30HL143859 United States
Welch FoundationI-1911 United States
CitationJournal: Mol Cell / Year: 2021
Title: Structural and mechanistic basis for protein glutamylation by the kinase fold.
Authors: Adam Osinski / Miles H Black / Krzysztof Pawłowski / Zhe Chen / Yang Li / Vincent S Tagliabracci /
Abstract: The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold, yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ...The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold, yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active-site Glu in SidE, resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.
History
DepositionApr 17, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 18, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 1, 2021Group: 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.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Nov 17, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
A: Calmodulin-dependent glutamylase SidJ
B: Calmodulin-2
C: Ubiquitinating/deubiquitinating enzyme SdeA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)214,3778
Polymers213,4343
Non-polymers9435
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Calmodulin-dependent glutamylase SidJ


Mass: 87374.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Legionella pneumophila (bacteria) / Gene: sidJ, lpg2155 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5ZTK6, Ligases
#2: Protein Calmodulin-2 /


Mass: 16939.623 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CALM2, CAM2, CAMB / Production host: Escherichia coli (E. coli) / References: UniProt: P0DP24
#3: Protein Ubiquitinating/deubiquitinating enzyme SdeA / Effector protein SdeA


Mass: 109120.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Legionella pneumophila (bacteria) / Gene: sdeA, lpg2157 / Production host: Escherichia coli (E. coli)
References: UniProt: Q5ZTK4, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, Transferases; Acyltransferases; Aminoacyltransferases, NAD+-protein-arginine ADP-ribosyltransferase

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Non-polymers , 4 types, 5 molecules

#4: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE / Adenosine monophosphate


Mass: 347.221 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#7: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION

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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

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of SidJ-SdeA-CaM reaction intermediate complexCOMPLEX#1-#30MULTIPLE SOURCES
2SidJ-SdeACOMPLEX#1, #31RECOMBINANT
3CalmodulinCOMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Legionella pneumophila (bacteria)446
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 6.5
Details: 25 mM Bis-tris pH 6.5, 100 mM NaCl, 1 mM TCEP, 2 mM MgCl2, 1 mM ATP
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 310154 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313258
ELECTRON MICROSCOPYf_angle_d0.44117903
ELECTRON MICROSCOPYf_dihedral_angle_d3.9791781
ELECTRON MICROSCOPYf_chiral_restr0.0361960
ELECTRON MICROSCOPYf_plane_restr0.0032348

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