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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 / bBAF complex / npBAF complex / nBAF complex / brahma complex / regulation of transepithelial transport / morphogenesis of a polarized epithelium / structural constituent of postsynaptic actin cytoskeleton / GBAF complex ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / bBAF complex / npBAF complex / nBAF complex / brahma complex / regulation of transepithelial transport / morphogenesis of a polarized epithelium / structural constituent of postsynaptic actin cytoskeleton / GBAF complex / Formation of annular gap junctions / Formation of the dystrophin-glycoprotein complex (DGC) / protein localization to adherens junction / Gap junction degradation / regulation of G0 to G1 transition / Folding of actin by CCT/TriC / dense body / Cell-extracellular matrix interactions / postsynaptic actin cytoskeleton / Tat protein binding / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / regulation of nucleotide-excision repair / regulation of double-strand break repair / adherens junction assembly / RHOF GTPase cycle / Adherens junctions interactions / apical protein localization / Sensory processing of sound by outer hair cells of the cochlea / tight junction / Interaction between L1 and Ankyrins / SWI/SNF complex / regulation of mitotic metaphase/anaphase transition / Sensory processing of sound by inner hair cells of the cochlea / positive regulation of T cell differentiation / regulation of norepinephrine uptake / apical junction complex / transporter regulator activity / positive regulation of double-strand break repair / maintenance of blood-brain barrier / nitric-oxide synthase binding / establishment or maintenance of cell polarity / cortical cytoskeleton / NuA4 histone acetyltransferase complex / positive regulation of stem cell population maintenance / Regulation of MITF-M-dependent genes involved in pigmentation / Recycling pathway of L1 / brush border / regulation of G1/S transition of mitotic cell cycle / kinesin binding / EPH-ephrin mediated repulsion of cells / negative regulation of cell differentiation / regulation of synaptic vesicle endocytosis / RHO GTPases Activate WASPs and WAVEs / positive regulation of myoblast differentiation / RHO GTPases activate IQGAPs / regulation of protein localization to plasma membrane / positive regulation of double-strand break repair via homologous recombination / cytoskeleton organization / EPHB-mediated forward signaling / substantia nigra development / axonogenesis / calyx of Held / nitric-oxide synthase regulator activity / DNA Damage Recognition in GG-NER / Translocation of SLC2A4 (GLUT4) to the plasma membrane / adherens junction / actin filament / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / FCGR3A-mediated phagocytosis / positive regulation of cell differentiation / cell motility / RHO GTPases Activate Formins / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / kinetochore / B-WICH complex positively regulates rRNA expression / Regulation of actin dynamics for phagocytic cup formation / structural constituent of cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / tau protein binding / platelet aggregation / VEGFA-VEGFR2 Pathway / Schaffer collateral - CA1 synapse / nuclear matrix / cytoplasmic ribonucleoprotein granule / UCH proteinases / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / cell-cell junction / Signaling by BRAF and RAF1 fusions / Clathrin-mediated endocytosis / nucleosome / actin cytoskeleton / presynapse / lamellipodium / HATs acetylate histones / Factors involved in megakaryocyte development and platelet production
Similarity search - Function
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

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)
Voxel sizeX=Y=Z: 0.822 Å
Density
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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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

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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

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