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- PDB-7p1g: Structure of the P. aeruginosa ExoY-F-actin complex -

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Basic information

Entry
Database: PDB / ID: 7p1g
TitleStructure of the P. aeruginosa ExoY-F-actin complex
Components
  • Actin, alpha skeletal muscle
  • Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY
  • Phalloidin
KeywordsTOXIN / Bacterial toxin / F-actin
Function / homology
Function and homology information


calcium- and calmodulin-responsive adenylate cyclase activity / cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / detection of maltose stimulus / actin filament bundle / maltose transport complex / troponin I binding / filamentous actin / mesenchyme migration ...calcium- and calmodulin-responsive adenylate cyclase activity / cytoskeletal motor activator activity / myosin heavy chain binding / tropomyosin binding / detection of maltose stimulus / actin filament bundle / maltose transport complex / troponin I binding / filamentous actin / mesenchyme migration / carbohydrate transport / skeletal muscle myofibril / actin filament bundle assembly / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / outer membrane-bounded periplasmic space / cell body / periplasmic space / hydrolase activity / protein domain specific binding / calcium ion binding / DNA damage response / positive regulation of gene expression / magnesium ion binding / extracellular region / ATP binding / identical protein binding / membrane / cytoplasm
Similarity search - Function
Anthrax toxin, edema factor, central / Anthrax toxin, edema factor, C-terminal / Anthrax toxin, edema factor, central domain superfamily / Anthrax toxin LF subunit / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / 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 ...Anthrax toxin, edema factor, central / Anthrax toxin, edema factor, C-terminal / Anthrax toxin, edema factor, central domain superfamily / Anthrax toxin LF subunit / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / 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. / Bacterial extracellular solute-binding protein / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / 3'-DEOXY-GUANOSINE-5'-TRIPHOSPHATE / PHOSPHATE ION / Maltose/maltodextrin-binding periplasmic protein / Actin, alpha skeletal muscle / Adenylate cyclase ExoY
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Pseudomonas aeruginosa PAO1 (bacteria)
Oryctolagus cuniculus (rabbit)
Amanita phalloides (death cap)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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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Structure visualization

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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
M: Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY
C: Actin, alpha skeletal muscle
H: Phalloidin
K: Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY
A: Actin, alpha skeletal muscle
F: Phalloidin
L: Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY
B: Actin, alpha skeletal muscle
G: Phalloidin
N: Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY
D: Actin, alpha skeletal muscle
I: Phalloidin
O: Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY
E: Actin, alpha skeletal muscle
J: Phalloidin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)644,62840
Polymers639,23815
Non-polymers5,39025
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 10 molecules MKLNOCABDE

#1: Protein
Maltose/maltodextrin-binding periplasmic protein,Adenylate cyclase ExoY / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 84941.797 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria), (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria)
Gene: malE, b4034, JW3994, exoY, PA2191 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): RIPL / References: UniProt: P0AEX9, UniProt: Q9I1S4
#2: Protein
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 42096.953 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P68135

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Protein/peptide , 1 types, 5 molecules HFGIJ

#3: Protein/peptide
Phalloidin


Mass: 808.899 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) Amanita phalloides (death cap)

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Non-polymers , 4 types, 25 molecules

#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-GH3 / 3'-DEOXY-GUANOSINE-5'-TRIPHOSPHATE


Type: RNA linking / Mass: 507.181 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O13P3
#6: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#7: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: PO4

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The complex of P. aeruginosa ExoY with F-actin / 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: 3500 nm / Nominal defocus min: 400 nm
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 93 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12437

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Processing

EM software
IDNameVersionCategory
2EPU1.8image acquisition
4CTFFINDCTF correction
7iMODFITmodel fitting
9PHENIXmodel refinement
10SPHIREinitial Euler assignment
11SPHIREfinal Euler assignment
12RELION3classification
13SPHIRE3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2249589
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1535755 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
17AD9

7ad9

B1
25XNW

5xnw

A1

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