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- EMDB-44118: Actin-bound Legionella pneumophila AMPylase LnaB with AMPylated c... -

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Basic information

Entry
Database: EMDB / ID: EMD-44118
TitleActin-bound Legionella pneumophila AMPylase LnaB with AMPylated catalytic histidine
Map data
Sample
  • Complex: Actin-bound Legionella pneumophila AMPylase LnaB with AMPylated catalytic histidine
    • Complex: LnaB
      • Protein or peptide: Type IV secretion protein Dot
    • Complex: Actin
      • Protein or peptide: Actin, cytoplasmic 1
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: CALCIUM ION
  • Ligand: LATRUNCULIN B
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
KeywordsAMPylase / antitoxin / actin / ubiquitin / TRANSFERASE
Function / homology
Function and homology information


positive regulation of norepinephrine uptake / cellular response to cytochalasin B / Formation of the embryonic stem cell BAF (esBAF) complex / regulation of transepithelial transport / Formation of the canonical BAF (cBAF) complex / morphogenesis of a polarized epithelium / Formation of annular gap junctions / Formation of the dystrophin-glycoprotein complex (DGC) / structural constituent of postsynaptic actin cytoskeleton / Formation of the polybromo-BAF (pBAF) complex ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / Formation of the embryonic stem cell BAF (esBAF) complex / regulation of transepithelial transport / Formation of the canonical BAF (cBAF) complex / morphogenesis of a polarized epithelium / Formation of annular gap junctions / Formation of the dystrophin-glycoprotein complex (DGC) / structural constituent of postsynaptic actin cytoskeleton / Formation of the polybromo-BAF (pBAF) complex / Gap junction degradation / GBP-mediated host defense / Formation of neuronal progenitor and neuronal BAF (npBAF and nBAF) / protein localization to adherens junction / Formation of the non-canonical BAF (ncBAF) complex / Cell-extracellular matrix interactions / regulation of G0 to G1 transition / dense body / Folding of actin by CCT/TriC / Tat protein binding / postsynaptic actin cytoskeleton / Regulation of CDH1 Function / apical protein localization / Prefoldin mediated transfer of substrate to CCT/TriC / Adherens junctions interactions / regulation of double-strand break repair / microtubule nucleation / adherens junction assembly / RHOF GTPase cycle / Sensory processing of sound by outer hair cells of the cochlea / tight junction / Sensory processing of sound by inner hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / positive regulation of T cell differentiation / maintenance of blood-brain barrier / Interaction between L1 and Ankyrins / apical junction complex / regulation of nucleotide-excision repair / positive regulation of stem cell population maintenance / NuA4 histone acetyltransferase complex / regulation of norepinephrine uptake / transporter regulator activity / Recycling pathway of L1 / cortical cytoskeleton / positive regulation of double-strand break repair / Regulation of MITF-M-dependent genes involved in pigmentation / negative regulation of cell differentiation / establishment or maintenance of cell polarity / nitric-oxide synthase binding / brush border / EPH-ephrin mediated repulsion of cells / positive regulation of myoblast differentiation / regulation of synaptic vesicle endocytosis / RHO GTPases Activate WASPs and WAVEs / kinesin binding / regulation of protein localization to plasma membrane / RHO GTPases activate IQGAPs / positive regulation of double-strand break repair via homologous recombination / regulation of G1/S transition of mitotic cell cycle / axonogenesis / cytoskeleton organization / EPHB-mediated forward signaling / substantia nigra development / calyx of Held / cell motility / nitric-oxide synthase regulator activity / FCGR3A-mediated phagocytosis / Translocation of SLC2A4 (GLUT4) to the plasma membrane / actin filament / positive regulation of cell differentiation / adherens junction / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / RHO GTPases Activate Formins / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / Regulation of actin dynamics for phagocytic cup formation / platelet aggregation / VEGFA-VEGFR2 Pathway / B-WICH complex positively regulates rRNA expression / tau protein binding / Schaffer collateral - CA1 synapse / DNA Damage Recognition in GG-NER / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / kinetochore / nuclear matrix / Signaling by RAF1 mutants / cell-cell junction / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / actin cytoskeleton / Signaling by BRAF and RAF1 fusions / UCH proteinases / lamellipodium / presynapse / HATs acetylate histones / Clathrin-mediated endocytosis / Factors involved in megakaryocyte development and platelet production
Similarity search - Function
Dot/Icm T4SS effector Ceg14/SidL / : / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Actin, cytoplasmic 1 / Type IV secretion protein Dot
Similarity search - Component
Biological speciesLegionella pneumophila subsp. pneumophila str. Philadelphia 1 (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsZhang Z / Das C
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21AI171709 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM126296 United States
CitationJournal: J Mol Biol / Year: 2025
Title: Cryo-EM Detection of AMPylated Histidine Implies Covalent Catalysis in AMPylation Mediated by a Bacterial Effector.
Authors: Zhengrui Zhang / Rishi Patel / Zhao-Qing Luo / Chittaranjan Das /
Abstract: AMPylation is a post-translational modification (PTM) whereby adenosine monophosphate (AMP) from adenosine triphosphate (ATP) is transferred onto protein hydroxyl groups of serine, threonine, or ...AMPylation is a post-translational modification (PTM) whereby adenosine monophosphate (AMP) from adenosine triphosphate (ATP) is transferred onto protein hydroxyl groups of serine, threonine, or tyrosine. Recently, an actin-dependent AMPylase namely LnaB from the bacterial pathogen Legionella pneumophila was found to AMPylate phosphate groups of phosphoribosylated ubiquitin and Src family kinases. LnaB represents an evolutionarily distinct family of AMPylases with conserved active site Ser-His-Glu residues. Here, we capture the structure of the LnaB-actin complex in a putative intermediate state via single-particle cryogenic electron microscopy (cryo-EM) and find that the catalytic histidine of LnaB is covalently attached to AMP through a phosphoramidate linkage at the Nδ1 atom. This observation provides direct structural evidence of histidine AMPylation as a PTM and implies the possibility of covalent catalysis in LnaB-mediated AMPylation, a mechanism distinct from known AMPylases. Subsequent biochemical studies confirm the observed AMP binding site and provide additional insights into the catalytic properties of LnaB. Together, our work highlights the power of cryo-EM in capturing labile PTMs and transient species during enzymatic reactions, while opening new avenues of mechanistic investigation into the LnaB family.
History
DepositionMar 18, 2024-
Header (metadata) releaseJan 8, 2025-
Map releaseJan 8, 2025-
UpdateJan 8, 2025-
Current statusJan 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44118.png

Downloads & links

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Map

FileDownload / File: emd_44118.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 256 pix.
= 210.432 Å		0.82 Å/pix.
x 256 pix.
= 210.432 Å		0.82 Å/pix.
x 256 pix.
= 210.432 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-1.2043028 - 1.6041691
Average (Standard dev.)0.00044130848 (±0.03602745)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 210.432 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44118_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_44118_half_map_2.map
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Histogram (log scale)

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Sample components

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Entire : Actin-bound Legionella pneumophila AMPylase LnaB with AMPylated c...

EntireName: Actin-bound Legionella pneumophila AMPylase LnaB with AMPylated catalytic histidine
Components
  • Complex: Actin-bound Legionella pneumophila AMPylase LnaB with AMPylated catalytic histidine
    • Complex: LnaB
      • Protein or peptide: Type IV secretion protein Dot
    • Complex: Actin
      • Protein or peptide: Actin, cytoplasmic 1
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: CALCIUM ION
  • Ligand: LATRUNCULIN B
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Actin-bound Legionella pneumophila AMPylase LnaB with AMPylated c...

SupramoleculeName: Actin-bound Legionella pneumophila AMPylase LnaB with AMPylated catalytic histidine
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: LnaB

SupramoleculeName: LnaB / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Legionella pneumophila subsp. pneumophila str. Philadelphia 1 (bacteria)

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Supramolecule #3: Actin

SupramoleculeName: Actin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Type IV secretion protein Dot

MacromoleculeName: Type IV secretion protein Dot / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Legionella pneumophila subsp. pneumophila str. Philadelphia 1 (bacteria)
Molecular weightTheoretical: 58.642035 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSYQKIELSI SLSLDSPKLD ESDFILLSVK YLEKSLGKKK EFSGFFEDIE KLYFKQNYKE AIEKILDFCK KNESLLSEQV VQRLAEVAP RLKSNPKDNE SRRLYETLYA DHLESVIKQE SDLSVFNELR DSYNAVKPEY AVTHETEIKT LDEAKQFILS F VMLNDNVE ...String:
MSYQKIELSI SLSLDSPKLD ESDFILLSVK YLEKSLGKKK EFSGFFEDIE KLYFKQNYKE AIEKILDFCK KNESLLSEQV VQRLAEVAP RLKSNPKDNE SRRLYETLYA DHLESVIKQE SDLSVFNELR DSYNAVKPEY AVTHETEIKT LDEAKQFILS F VMLNDNVE LPLKAQSERY PKKDRSREEL GNTPSANPGI MKPNSPNFTD NLVPVRDVPK IAINEKVAGG YSKTKPTTPF VA SLAGTTY SLMVVLTDYI EKHKTDKDIE KKVNQIINLW ISSYIKEGYH SYSEVVDVLT EPFLQSIFDK ANIKLNYGVL DDT HAEFRK AQDYVFGLTI QSAMHHELQE RFKNKEKLQE EVKNFESGLN KLNQNVENTG RHREALNKLN EVFQDWSAGK KSYD AFKSE SNQLIHDIES EEQKSNRGLG SMLKNLGNYL LYLITLRFLK EDYPKPSSPV TMLVTELRGT LEQIDHFHKT SLSLK KATQ PDPKNTSSSD ETADVRKSAH HHHHH

UniProtKB: Type IV secretion protein Dot

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Macromolecule #2: Actin, cytoplasmic 1

MacromoleculeName: Actin, cytoplasmic 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.78266 KDa
SequenceString: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIVTNWD DMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSGDGV T HTVPIYEG ...String:
MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IEHGIVTNWD DMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSGDGV T HTVPIYEG YALPHAILRL DLAGRDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSLEK SY ELPDGQV ITIGNERFRC PEALFQPSFL GMESCGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKEI TAL APSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF

UniProtKB: Actin, cytoplasmic 1

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Macromolecule #3: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: LATRUNCULIN B

MacromoleculeName: LATRUNCULIN B / type: ligand / ID: 5 / Number of copies: 1 / Formula: LAB
Molecular weightTheoretical: 395.513 Da
Chemical component information

ChemComp-LAB:
LATRUNCULIN B / toxin*YM

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Macromolecule #6: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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

Concentration1.0 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 59.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 536510
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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