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- PDB-6s5t: Legionella pneumophila SidJ-Human calmodulin complex -

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Basic information

Entry
Database: PDB / ID: 6s5t
TitleLegionella pneumophila SidJ-Human calmodulin complex
Components
  • Calmodulin-2
  • SidJ
KeywordsTRANSFERASE / Bacterial glutamylase / pseudo kinase / calmodulin-dependent / Legionella / SidJ / SdeA / serine ubiquitination
Function / homology
Function and homology information


Ligases / negative regulation of calcium ion transmembrane transporter 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 phosphatase activator activity / positive regulation of cyclic-nucleotide phosphodiesterase activity / positive regulation of phosphoprotein phosphatase activity / ligase activity ...Ligases / negative regulation of calcium ion transmembrane transporter 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 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 / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / cysteine-type peptidase activity / 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 / 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 / G protein-coupled receptor signaling pathway / nucleotide binding / centrosome / calcium ion binding / protein kinase binding / protein-containing complex / proteolysis / membrane / metal ion binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Calmodulin-2 / Calmodulin-dependent glutamylase SidJ
Similarity search - Component
Biological speciesLegionella pneumophila (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.15 Å
AuthorsBhogaraju, S. / Galej, W.P. / Pfleiderer, M.M. / Adams, M.
Funding support Germany, 3items
OrganizationGrant numberCountry
German Research FoundationSFB 1177 Germany
German Research FoundationEXC115 Germany
European Research Council742720
CitationJournal: Nature / Year: 2019
Title: Inhibition of bacterial ubiquitin ligases by SidJ-calmodulin catalysed glutamylation.
Authors: Sagar Bhogaraju / Florian Bonn / Rukmini Mukherjee / Michael Adams / Moritz M Pfleiderer / Wojciech P Galej / Vigor Matkovic / Jaime Lopez-Mosqueda / Sissy Kalayil / Donghyuk Shin / Ivan Dikic /
Abstract: The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease. ...The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease. SidE enzymes share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes the toxicity of these enzymes in yeast and mammalian cells, through a mechanism that is currently unknown. Deletion of SidJ leads to a substantial defect in the growth of Legionella in both its natural hosts (amoebae) and in mouse macrophages. Here we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADP ribosyl transferase domain of the SdeA, thus blocking the ubiquitin ligase activity of SdeA. The glutamylation activity of SidJ requires interaction with the eukaryotic-specific co-factor calmodulin, and can be regulated by intracellular changes in Ca concentrations. The cryo-electron microscopy structure of SidJ in complex with human apo-calmodulin revealed the architecture of this heterodimeric glutamylase. We show that, in cells infected with L. pneumophila, SidJ mediates the glutamylation of SidE enzymes on the surface of vacuoles that contain Legionella. We used quantitative proteomics to uncover multiple host proteins as putative targets of SidJ-mediated glutamylation. Our study reveals the mechanism by which SidE ligases are inhibited by a SidJ-calmodulin glutamylase, and opens avenues for exploring an understudied protein modification (glutamylation) in eukaryotes.
History
DepositionJul 2, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 24, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Aug 28, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: SidJ
B: Calmodulin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)117,6973
Polymers117,1912
Non-polymers5061
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4340 Å2
ΔGint-15 kcal/mol
Surface area41300 Å2
MethodPISA

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Components

#1: Protein SidJ


Mass: 100338.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Legionella pneumophila (bacteria) / Gene: lpg2155 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5ZTK6
#2: Protein Calmodulin-2 /


Mass: 16852.545 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: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
Has ligand of interestN

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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
1Legionella pneumophila SidJ - human calmodulin complexCOMPLEX#1-#20MULTIPLE SOURCES
2SidJCOMPLEX#11RECOMBINANT
3calmodulinCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental 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: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Recombinant Legionella pneumophila SidJ -human calmodulin complex
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1Warp1.6particle selection
2EPU2image acquisition
4CTFFIND4.15CTF correction
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19851 / Symmetry type: POINT

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