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- EMDB-13583: Structure of SidJ/CaM bound to SdeA in pre-glutamylation state -

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Basic information

Entry
Database: EMDB / ID: EMD-13583
TitleStructure of SidJ/CaM bound to SdeA in pre-glutamylation state
Map data
Sample
  • Complex: SidJ/CaM-SdeA
    • Protein or peptide: Ubiquitinating/deubiquitinating enzyme SdeA
    • Protein or peptide: Calmodulin-dependent glutamylase SidJ
    • Protein or peptide: Calmodulin
  • Ligand: MAGNESIUM ION
  • Ligand: CALCIUM ION
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 / K63-linked deubiquitinase activity / positive regulation of cyclic-nucleotide phosphodiesterase 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 / K63-linked deubiquitinase activity / positive regulation of cyclic-nucleotide phosphodiesterase 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 / protein deubiquitination / negative regulation of peptidyl-threonine phosphorylation / protein phosphatase activator activity / ligase activity / positive regulation of phosphoprotein phosphatase activity / adenylate cyclase binding / catalytic complex / detection of calcium ion / regulation of cardiac muscle contraction / negative regulation of ryanodine-sensitive calcium-release channel activity / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / cysteine-type peptidase activity / positive regulation of protein dephosphorylation / regulation of calcium-mediated signaling / titin binding / positive regulation of protein autophosphorylation / voltage-gated potassium channel complex / sperm midpiece / calcium channel complex / substantia nigra development / adenylate cyclase activator activity / regulation of heart rate / nucleotidyltransferase activity / sarcomere / protein serine/threonine kinase activator activity / positive regulation of peptidyl-threonine phosphorylation / regulation of cytokinesis / positive regulation of protein serine/threonine kinase activity / spindle microtubule / spindle pole / response to calcium ion / G2/M transition of mitotic cell cycle / calcium-dependent protein binding / host cell / 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 / nucleus / metal ion binding / 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
Calmodulin-2 / Ubiquitinating/deubiquitinating enzyme SdeA / Calmodulin-dependent glutamylase SidJ
Similarity search - Component
Biological speciesLegionella pneumophila (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsAdams M / Bhogaraju S
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Union (EU)European Union
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis for protein glutamylation by the Legionella pseudokinase SidJ.
Authors: Michael Adams / Rahul Sharma / Thomas Colby / Felix Weis / Ivan Matic / Sagar Bhogaraju /
Abstract: Legionella pneumophila (LP) avoids phagocytosis by secreting nearly 300 effector proteins into the host cytosol. SidE family of effectors (SdeA, SdeB, SdeC and SidE) employ phosphoribosyl ...Legionella pneumophila (LP) avoids phagocytosis by secreting nearly 300 effector proteins into the host cytosol. SidE family of effectors (SdeA, SdeB, SdeC and SidE) employ phosphoribosyl ubiquitination to target multiple host Rab GTPases and innate immune factors. To suppress the deleterious toxicity of SidE enzymes in a timely manner, LP employs a metaeffector named SidJ. Upon activation by host Calmodulin (CaM), SidJ executes an ATP-dependent glutamylation to modify the catalytic residue Glu860 in the mono-ADP-ribosyl transferase (mART) domain of SdeA. SidJ is a unique glutamylase that adopts a kinase-like fold but contains two nucleotide-binding pockets. There is a lack of consensus about the substrate recognition and catalytic mechanism of SidJ. Here, we determined the cryo-EM structure of SidJ in complex with its substrate SdeA in two different states of catalysis. Our structures reveal that both phosphodiesterase (PDE) and mART domains of SdeA make extensive contacts with SidJ. In the pre-glutamylation state structure of the SidJ-SdeA complex, adenylylated E860 of SdeA is inserted into the non-canonical (migrated) nucleotide-binding pocket of SidJ. Structure-based mutational analysis indicates that SidJ employs its migrated pocket for the glutamylation of SdeA. Finally, using mass spectrometry, we identified several transient autoAMPylation sites close to both the catalytic pockets of SidJ. Our data provide unique insights into the substrate recognition and the mechanism of protein glutamylation by the pseudokinase SidJ.
History
DepositionSep 14, 2021-
Header (metadata) releaseOct 27, 2021-
Map releaseOct 27, 2021-
UpdateNov 17, 2021-
Current statusNov 17, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ppo
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13583.png

Downloads & links

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Map

FileDownload / File: emd_13583.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.008 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.16462716 - 0.3079889
Average (Standard dev.)7.820398e-05 (±0.005332953)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 282.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0081.0081.008
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z282.240282.240282.240
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.1650.3080.000

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Supplemental data

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Mask #1

Fileemd_13583_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SidJ/CaM-SdeA

EntireName: SidJ/CaM-SdeA
Components
  • Complex: SidJ/CaM-SdeA
    • Protein or peptide: Ubiquitinating/deubiquitinating enzyme SdeA
    • Protein or peptide: Calmodulin-dependent glutamylase SidJ
    • Protein or peptide: Calmodulin
  • Ligand: MAGNESIUM ION
  • Ligand: CALCIUM ION

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Supramolecule #1: SidJ/CaM-SdeA

SupramoleculeName: SidJ/CaM-SdeA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Legionella pneumophila (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Ubiquitinating/deubiquitinating enzyme SdeA

MacromoleculeName: Ubiquitinating/deubiquitinating enzyme SdeA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
Source (natural)Organism: Legionella pneumophila (bacteria)
Molecular weightTheoretical: 111.200375 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HHHHHHSAGL EVLFQGPMVG FSLYTDDTVK AAAQYAYDNY LGKPYTGSVE SAPANFGGRM VYRQHHGLSH TLRTMAYAEL IVEEARKAK LRGETLGKFK DGRTIADVTP QELKKIMIAQ AFFVAGRDDE ASDAKNYQKY HEQSRDAFLK YVKDNESTLI P DVFKDQED ...String:
HHHHHHSAGL EVLFQGPMVG FSLYTDDTVK AAAQYAYDNY LGKPYTGSVE SAPANFGGRM VYRQHHGLSH TLRTMAYAEL IVEEARKAK LRGETLGKFK DGRTIADVTP QELKKIMIAQ AFFVAGRDDE ASDAKNYQKY HEQSRDAFLK YVKDNESTLI P DVFKDQED VNFYARVIED KSHDWESTPA HVLINQGHMV DLVRVKQPPE SFLQRYFSSM QRWIGSQATE AVFGIQRQFF HA TYEVVAG FDSDNKEPHL VVSGLGRYVI GEDGQPIREA PKKGQKEGDL KVFPQTYKLK ENERLMRVDE FLKLPEIQNT FPG SGKHLQ GGMPGMNEMD YWNRLNSLNR ARCENDVDFC LKQLQTAHDK AKIEPIKQAF QSSKGKERRQ PNVDEIAAAR IIQQ ILANP DCIHDDHVLI NGQKLEQQFF RDLLAKCEMA VVGSLLNDTD IGNIDTLMRH EKDTEFHSTN PEAVPVKIGE YWIND QRIN NSSGNITQKK HDLIFLMQND AWYFSRVNAI AQNRDKGSTF KEVLITTLMT PLTSKALVDT SQAKPPTRLF RGLNLS EEF TKGLIDQANA MIANTTERLF TDHSPEAFKQ IKLNDLSKMS GRTNASTTTE IKLVKETWDS NVIFEMLDPD GLLHSKQ VG RHGEGT(86N)SEF SVYLPEDVAL VPVKVTLDGK TQKGENRYVF TFVAVKSPDF TPRHESGYAV EPFLRMQAAK LAEV KSSIE KAQRAPDLET IFNLQNEVEA VQYSHLSTGY KNFLKNTVGP VLENSLSGLM ESDTDTLSKA LAAFPSDTQW SAFNF EEAR QAKRQMDAIK QMVGNKVVLD ALTQCQDALE KQNIAGALDA LKKIPSEKEM GTIRRELREQ IQSARQELES LQRAVV TPV VTDEKKVRER YDALIENTSK KITELETGKL PNLDAVKKGI SNLSNLKQEV TVLRNEKIRM HVGTDKVDFS DVEKLEQ QI QVIDTKLADA YLLEVTKQIS A

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Macromolecule #2: Calmodulin-dependent glutamylase SidJ

MacromoleculeName: Calmodulin-dependent glutamylase SidJ / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: Ligases
Source (natural)Organism: Legionella pneumophila (bacteria)
Molecular weightTheoretical: 91.826992 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HHHHHHSAGL EVLFQGPMVK QYYFARRGET STHDTSLPPP VKVLSGRSIP LKEIPFETTR NELVQIYLTS VDQLIKSNKL NSIPSQQIA SHYLFLRSLA NSETDGIKKN QILSLAKPLG IYLASKEPHV WKTINELIEK SEYPIIHYLK NNRAHSNFML A LIHEYHKE ...String:
HHHHHHSAGL EVLFQGPMVK QYYFARRGET STHDTSLPPP VKVLSGRSIP LKEIPFETTR NELVQIYLTS VDQLIKSNKL NSIPSQQIA SHYLFLRSLA NSETDGIKKN QILSLAKPLG IYLASKEPHV WKTINELIEK SEYPIIHYLK NNRAHSNFML A LIHEYHKE PLTKNQSAFV QKFRDSSVFL FPNPIYTAWL AHSYDEDSSF NPMFRERLST NFYHSTLTDN LLLRTEPKEV TL SSEHHYK KEKGPIDSSF RYQMSSDRLL RIQGRTLLFS TPQNDVVAVK VQKRGEPKST LEEEFQMADY LLKHQSRLDV YSK LPQPLG QYSVKKSEIL EISRGSLDFE RFKTLIGDSK DLEVYVYKAP LTYFTYLHDK NQDLEDLTAS VKTNVHDLFV LLRE GIMFP QLADIFHTHF GEDEREDKGR YQALVQLLNV LQFQLGRIDK WQKAVEYVNL RSSGLADLGD SLPITSLFTS SDFTK HYFS ALLTGGYHPT FFDKSSGTAN SLFTGKRRLF GNYLYLNTIA EYLLVIQLTL GSYGDKVTRD MMDKPKKEAV WRELAN VMF TSCAEAIHIM TGIPQSRALT LLKQRANIEK HFRQTQFWMT PDYSKLDEDT LQMEQYSIYS GEPEYEFTDK LVSGVGL SV DGTHQDLGGY NRESPLRELE KLLYATVTLI EGTMQLDKEF FKQLQQVEKI LSGEIKTDAN SCFEAVAQLL DLARPRCH F QKRLVLSYYE EAKLKYPSAP TDAYDSRFQV VAKTNAAITI QRFWRETRKN LSENSDIESE KPESERTTDK RLK

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Macromolecule #3: Calmodulin

MacromoleculeName: Calmodulin / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 19.066006 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
HHHHHHSSGL EVLFQGPHMM ADQLTEEQIA EFKEAFSLFD KDGDGTITTK ELGTVMRSLG QNPTEAELQD MINEVDADGN GTIDFPEFL TMMARKMKDT DSEEEIREAF RVFDKDGNGY ISAAELRHVM TNLGEKLTDE EVDEMIREAD IDGDGQVNYE E FVQMMTAK

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.45 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 140022
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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