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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 / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / synapse maturation / modification of postsynaptic actin cytoskeleton / regulation of transepithelial transport / negative regulation of actin filament bundle assembly / adenyl-nucleotide exchange factor activity / morphogenesis of a polarized epithelium / bBAF complex ...calcium- and calmodulin-responsive adenylate cyclase activity / positive regulation of norepinephrine uptake / cellular response to cytochalasin B / synapse maturation / modification of postsynaptic actin cytoskeleton / regulation of transepithelial transport / negative regulation of actin filament bundle assembly / adenyl-nucleotide exchange factor activity / morphogenesis of a polarized epithelium / bBAF complex / postsynaptic actin cytoskeleton organization / protein localization to adherens junction / postsynaptic actin cytoskeleton / npBAF complex / Tat protein binding / brahma complex / structural constituent of postsynaptic actin cytoskeleton / nBAF complex / GBAF complex / positive regulation of actin filament bundle assembly / negative regulation of actin filament polymerization / regulation of G0 to G1 transition / dense body / Formation of annular gap junctions / Gap junction degradation / Signaling by ROBO receptors / Cell-extracellular matrix interactions / Folding of actin by CCT/TriC / regulation of actin filament polymerization / apical protein localization / regulation of double-strand break repair / regulation of nucleotide-excision repair / adherens junction assembly / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / RHOF GTPase cycle / Adherens junctions interactions / tight junction / positive regulation of ATP-dependent activity / PCP/CE pathway / proline-rich region binding / Sensory processing of sound by outer hair cells of the cochlea / regulation of norepinephrine uptake / positive regulation of ruffle assembly / regulation of mitotic metaphase/anaphase transition / Interaction between L1 and Ankyrins / Sensory processing of sound by inner hair cells of the cochlea / SWI/SNF complex / positive regulation of double-strand break repair / regulation of synaptic vesicle endocytosis / positive regulation of T cell differentiation / negative regulation of stress fiber assembly / host cell cytosol / apical junction complex / establishment or maintenance of cell polarity / regulation of cyclin-dependent protein serine/threonine kinase activity / cortical cytoskeleton / maintenance of blood-brain barrier / positive regulation of actin filament polymerization / positive regulation of stem cell population maintenance / detection of maltose stimulus / NuA4 histone acetyltransferase complex / maltose transport complex / nitric-oxide synthase binding / positive regulation of epithelial cell migration / Recycling pathway of L1 / regulation of G1/S transition of mitotic cell cycle / kinesin binding / carbohydrate transport / brush border / calyx of Held / negative regulation of cell differentiation / carbohydrate transmembrane transporter activity / positive regulation of double-strand break repair via homologous recombination / : / maltose binding / actin monomer binding / 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 / substantia nigra development / phosphatidylinositol-4,5-bisphosphate binding / phosphotyrosine residue binding / ATP-binding cassette (ABC) transporter complex / axonogenesis / cell chemotaxis / negative regulation of protein binding / neural tube closure / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / cell motility / actin filament / positive regulation of cell differentiation / regulation of transmembrane transporter activity
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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