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- PDB-7p1h: Structure of the V. vulnificus ExoY-G-actin-profilin complex -

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Basic information

Entry
Database: PDB / ID: 7p1h
TitleStructure of the V. vulnificus ExoY-G-actin-profilin complex
Components
  • Actin, cytoplasmic 1
  • Maltose/maltodextrin-binding periplasmic protein,RTX-toxin
  • Profilin-1
KeywordsTOXIN / Bacterial toxin / G-actin / profilin
Function / homology
Function and homology information


calcium- and calmodulin-responsive adenylate cyclase activity / synapse maturation / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / modification of postsynaptic actin cytoskeleton / negative regulation of actin filament bundle assembly / regulation of transepithelial transport / adenyl-nucleotide exchange factor activity / morphogenesis of a polarized epithelium / bBAF complex ...calcium- and calmodulin-responsive adenylate cyclase activity / synapse maturation / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / modification of postsynaptic actin cytoskeleton / negative regulation of actin filament bundle assembly / regulation of transepithelial transport / adenyl-nucleotide exchange factor activity / morphogenesis of a polarized epithelium / bBAF complex / postsynaptic actin cytoskeleton organization / npBAF complex / nBAF complex / protein localization to adherens junction / brahma complex / postsynaptic actin cytoskeleton / Tat protein binding / structural constituent of postsynaptic actin cytoskeleton / positive regulation of actin filament bundle assembly / negative regulation of actin filament polymerization / GBAF complex / dense body / Formation of annular gap junctions / regulation of G0 to G1 transition / Signaling by ROBO receptors / Gap junction degradation / regulation of actin filament polymerization / 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 / Regulation of MITF-M-dependent genes involved in pigmentation / positive regulation of ATP-dependent activity / Adherens junctions interactions / tight junction / PCP/CE pathway / proline-rich region binding / positive regulation of ruffle assembly / Sensory processing of sound by outer hair cells of the cochlea / regulation of norepinephrine uptake / Interaction between L1 and Ankyrins / Sensory processing of sound by inner hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / SWI/SNF complex / positive regulation of double-strand break repair / regulation of synaptic vesicle endocytosis / negative regulation of stress fiber assembly / positive regulation of T cell differentiation / apical junction complex / host cell cytosol / establishment or maintenance of cell polarity / regulation of cyclin-dependent protein serine/threonine kinase activity / positive regulation of actin filament polymerization / detection of maltose stimulus / maintenance of blood-brain barrier / cortical cytoskeleton / positive regulation of stem cell population maintenance / maltose transport complex / positive regulation of epithelial cell migration / NuA4 histone acetyltransferase complex / nitric-oxide synthase binding / regulation of G1/S transition of mitotic cell cycle / Recycling pathway of L1 / carbohydrate transport / kinesin binding / brush border / calyx of Held / negative regulation of cell differentiation / carbohydrate transmembrane transporter activity / : / actin monomer binding / maltose binding / positive regulation of double-strand break repair via homologous recombination / maltose transport / maltodextrin transmembrane transport / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / regulation of protein localization to plasma membrane / positive regulation of myoblast differentiation / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / EPHB-mediated forward signaling / phosphatidylinositol-4,5-bisphosphate binding / substantia nigra development / phosphotyrosine residue binding / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / axonogenesis / negative regulation of protein binding / neural tube closure / Translocation of SLC2A4 (GLUT4) to the plasma membrane / actin filament / cell motility / RHO GTPases Activate Formins / positive regulation of cell differentiation
Similarity search - Function
Peptidase C58, YopT-type domain / Yersinia/Haemophilus virulence surface antigen / Profilin1/2/3, vertebrate / Anthrax toxin, edema factor, central / Anthrax toxin, edema factor, C-terminal / Anthrax toxin, edema factor, central domain superfamily / Anthrax toxin LF subunit / : / Profilin conserved site / Profilin signature. ...Peptidase C58, YopT-type domain / Yersinia/Haemophilus virulence surface antigen / Profilin1/2/3, vertebrate / Anthrax toxin, edema factor, central / Anthrax toxin, edema factor, C-terminal / Anthrax toxin, edema factor, central domain superfamily / Anthrax toxin LF subunit / : / Profilin conserved site / Profilin signature. / Profilin / Profilin / Profilin / Profilin superfamily / Dermonecrotic/RTX toxin, membrane localization domain / Membrane Localization Domain / Serine aminopeptidase, S33 / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily / Peptidase C80 family / CGT/MARTX cysteine protease (CPD) domain profile. / Serine aminopeptidase, S33 / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Bacterial extracellular solute-binding protein / Actin / Actin family / Actin / ATPase, nucleotide binding domain / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / RTX-toxin / Profilin-1 / Maltose/maltodextrin-binding periplasmic protein / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Vibrio vulnificus (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBelyy, A. / Merino, F. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Commun / Year: 2021
Title: Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens.
Authors: Alexander Belyy / Felipe Merino / Undine Mechold / Stefan Raunser /
Abstract: Bacterial human pathogens secrete initially inactive nucleotidyl cyclases that become potent enzymes by binding to actin inside eukaryotic host cells. The underlying molecular mechanism of this ...Bacterial human pathogens secrete initially inactive nucleotidyl cyclases that become potent enzymes by binding to actin inside eukaryotic host cells. The underlying molecular mechanism of this activation is, however, unclear. Here, we report structures of ExoY from Pseudomonas aeruginosa and Vibrio vulnificus bound to their corresponding activators F-actin and profilin-G-actin. The structures reveal that in contrast to the apo-state, two flexible regions become ordered and interact strongly with actin. The specific stabilization of these regions results in an allosteric stabilization of the nucleotide binding pocket and thereby to an activation of the enzyme. Differences in the sequence and conformation of the actin-binding regions are responsible for the selective binding to either F- or G-actin. Other nucleotidyl cyclase toxins that bind to calmodulin rather than actin undergo a similar disordered-to-ordered transition during activation, suggesting that the allosteric activation-by-stabilization mechanism of ExoY is conserved in these enzymes, albeit the different activator.
History
DepositionJul 1, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 1, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Assembly

Deposited unit
A: Maltose/maltodextrin-binding periplasmic protein,RTX-toxin
B: Actin, cytoplasmic 1
P: Profilin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,4885
Polymers147,9413
Non-polymers5472
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5970 Å2
ΔGint-34 kcal/mol
Surface area39120 Å2
MethodPISA

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Components

#1: Protein Maltose/maltodextrin-binding periplasmic protein,RTX-toxin / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 91467.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria), (gene. exp.) Vibrio vulnificus (bacteria)
Strain: K12 / Gene: malE, b4034, JW3994 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 DE3 RIPL / References: UniProt: P0AEX9, UniProt: A0A0F6NGV0
#2: Protein Actin, cytoplasmic 1 / Beta-actin


Mass: 41402.242 Da / Num. of mol.: 1 / Mutation: C272A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACTB / Cell line (production host): BTI-Tnao38 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P60709
#3: Protein Profilin-1 / Epididymis tissue protein Li 184a / Profilin I


Mass: 15071.222 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PFN1 / Production host: Escherichia coli (E. coli) / References: UniProt: P07737
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#5: 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
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

ComponentName: Structure of the V. vulnificus ExoY-G-actin-profilin complex
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingAverage exposure time: 2 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6879

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
1crYOLOparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7ISOLDEmodel fitting
9SPHIREinitial Euler assignment
10SPHIREfinal Euler assignment
12SPHIRE3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1252333
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 342081 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16NBW

6nbw

A1
26NBW

6nbw

C1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0057219
ELECTRON MICROSCOPYf_angle_d1.0229791
ELECTRON MICROSCOPYf_dihedral_angle_d23.2182673
ELECTRON MICROSCOPYf_chiral_restr0.0521086
ELECTRON MICROSCOPYf_plane_restr0.0061254

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