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- PDB-9b2z: Actin-bound Legionella pneumophila AMPylase LnaB with AMPylated c... -

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Basic information

Entry
Database: PDB / ID: 9b2z
TitleActin-bound Legionella pneumophila AMPylase LnaB with AMPylated catalytic histidine
Components
  • Actin, cytoplasmic 1
  • Type IV secretion protein Dot
KeywordsTRANSFERASE / AMPylase / antitoxin / actin / ubiquitin
Function / homology
Function and homology information


positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / morphogenesis of a polarized epithelium / bBAF complex / postsynaptic actin cytoskeleton / npBAF complex / protein localization to adherens junction / nBAF complex / brahma complex ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / morphogenesis of a polarized epithelium / bBAF complex / postsynaptic actin cytoskeleton / npBAF complex / protein localization to adherens junction / nBAF complex / brahma complex / Tat protein binding / GBAF complex / structural constituent of postsynaptic actin cytoskeleton / Formation of annular gap junctions / regulation of G0 to G1 transition / Gap junction degradation / dense body / Cell-extracellular matrix interactions / Folding of actin by CCT/TriC / apical protein localization / regulation of double-strand break repair / adherens junction assembly / regulation of nucleotide-excision repair / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / RHOF GTPase cycle / tight junction / Adherens junctions interactions / Interaction between L1 and Ankyrins / Sensory processing of sound by outer hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / Sensory processing of sound by inner hair cells of the cochlea / SWI/SNF complex / regulation of norepinephrine uptake / positive regulation of double-strand break repair / apical junction complex / positive regulation of T cell differentiation / nitric-oxide synthase binding / regulation of synaptic vesicle endocytosis / establishment or maintenance of cell polarity / regulation of cyclin-dependent protein serine/threonine kinase activity / maintenance of blood-brain barrier / cortical cytoskeleton / positive regulation of stem cell population maintenance / NuA4 histone acetyltransferase complex / Regulation of MITF-M-dependent genes involved in pigmentation / brush border / regulation of G1/S transition of mitotic cell cycle / Recycling pathway of L1 / kinesin binding / negative regulation of cell differentiation / EPH-ephrin mediated repulsion of cells / regulation of protein localization to plasma membrane / RHO GTPases Activate WASPs and WAVEs / positive regulation of double-strand break repair via homologous recombination / RHO GTPases activate IQGAPs / positive regulation of myoblast differentiation / calyx of Held / EPHB-mediated forward signaling / substantia nigra development / axonogenesis / negative regulation of protein binding / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / Translocation of SLC2A4 (GLUT4) to the plasma membrane / actin filament / adherens junction / RHO GTPases Activate Formins / positive regulation of cell differentiation / FCGR3A-mediated phagocytosis / cell motility / regulation of transmembrane transporter activity / Signaling by high-kinase activity BRAF mutants / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA Damage Recognition in GG-NER / MAP2K and MAPK activation / tau protein binding / B-WICH complex positively regulates rRNA expression / Schaffer collateral - CA1 synapse / Regulation of actin dynamics for phagocytic cup formation / structural constituent of cytoskeleton / kinetochore / platelet aggregation / VEGFA-VEGFR2 Pathway / nuclear matrix / cytoplasmic ribonucleoprotein granule / 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 / UCH proteinases / Signaling by BRAF and RAF1 fusions / cell-cell junction / nucleosome / actin cytoskeleton / lamellipodium / presynapse / Clathrin-mediated endocytosis / HATs acetylate histones / Factors involved in megakaryocyte development and platelet production / regulation of apoptotic process
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
ADENOSINE MONOPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / LATRUNCULIN B / Actin, cytoplasmic 1 / Type IV secretion protein Dot
Similarity search - Component
Biological speciesLegionella pneumophila subsp. pneumophila str. Philadelphia 1 (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsZhang, Z. / Das, C.
Funding support United States, 2items
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: 2024
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 8, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Type IV secretion protein Dot
B: Actin, cytoplasmic 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,7156
Polymers100,4252
Non-polymers1,2904
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Type IV secretion protein Dot


Mass: 58642.035 Da / Num. of mol.: 1 / Mutation: S261A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Legionella pneumophila subsp. pneumophila str. Philadelphia 1 (bacteria)
Gene: lpg2527 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5ZSJ0
#2: Protein Actin, cytoplasmic 1 / Beta-actin


Mass: 41782.660 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P60709

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Non-polymers , 4 types, 4 molecules

#3: Chemical ChemComp-AMP / ADENOSINE MONOPHOSPHATE


Mass: 347.221 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP*YM
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-LAB / LATRUNCULIN B


Mass: 395.513 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H29NO5S / Feature type: SUBJECT OF INVESTIGATION / Comment: toxin*YM
#6: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestY
Has protein modificationY

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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
1Actin-bound Legionella pneumophila AMPylase LnaB with AMPylated catalytic histidineCOMPLEX#1-#20RECOMBINANT
2LnaBCOMPLEX#11RECOMBINANT
3ActinCOMPLEX#21NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Legionella pneumophila subsp. pneumophila str. Philadelphia 1 (bacteria)272624
23Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 59.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.18.2_3874: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 536510 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035626
ELECTRON MICROSCOPYf_angle_d0.6297655
ELECTRON MICROSCOPYf_dihedral_angle_d12.544803
ELECTRON MICROSCOPYf_chiral_restr0.046882
ELECTRON MICROSCOPYf_plane_restr0.004966

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