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- PDB-8ds6: Structure of the PEAK3 pseudokinase homodimer -

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Basic information

Entry
Database: PDB / ID: 8ds6
TitleStructure of the PEAK3 pseudokinase homodimer
ComponentsProtein PEAK3
KeywordsSIGNALING PROTEIN / complex / pseudokinase / kinase / adapter
Function / homologyregulation of actin cytoskeleton organization / : / actin cytoskeleton / regulation of cell shape / protein kinase activity / focal adhesion / Protein PEAK3
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsTorosyan, H. / Paul, M. / Jura, N. / Verba, K.A.
Funding support United States, 3items
OrganizationGrant numberCountry
Other governmentQBI Independent Research Fellowship
National Institutes of Health/National Cancer Institute (NIH/NCI)U54 CA209891 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM139636 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural insights into regulation of the PEAK3 pseudokinase scaffold by 14-3-3.
Authors: Hayarpi Torosyan / Michael D Paul / Antoine Forget / Megan Lo / Devan Diwanji / Krzysztof Pawłowski / Nevan J Krogan / Natalia Jura / Kliment A Verba /
Abstract: PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK ...PEAK pseudokinases are molecular scaffolds which dimerize to regulate cell migration, morphology, and proliferation, as well as cancer progression. The mechanistic role dimerization plays in PEAK scaffolding remains unclear, as there are no structures of PEAKs in complex with their interactors. Here, we report the cryo-EM structure of dimeric PEAK3 in complex with an endogenous 14-3-3 heterodimer. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the SHED domain of the PEAK3 dimer. The binding interface contains a canonical phosphosite-dependent primary interaction and a unique secondary interaction not observed in previous structures of 14-3-3/client complexes. Additionally, we show that PKD regulates PEAK3/14-3-3 binding, which when prevented leads to PEAK3 nuclear enrichment and distinct protein-protein interactions. Altogether, our data demonstrate that PEAK3 dimerization forms an unusual secondary interface for 14-3-3 binding, facilitating 14-3-3 regulation of PEAK3 localization and interactome diversity.
History
DepositionJul 21, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protein PEAK3
B: Protein PEAK3


Theoretical massNumber of molelcules
Total (without water)104,7142
Polymers104,7142
Non-polymers00
Water00
1


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

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Components

#1: Protein Protein PEAK3


Mass: 52357.031 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PEAK3, C19orf35 / Production host: Homo sapiens (human) / References: UniProt: Q6ZS72

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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: PEAK3 pseudokinase homodimer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.104580 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Details: A final concentration of 0.1% of Octyl-beta-Glucoside (C14H28O6) was added to the sample before freezing.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris BaseNH2C(CH2OH)31
2150 mMSodium ChlorideNaCl1
32 mMDithiothreitolC4H10O2S21
42 mMMagnesium ChlorideMgCl21
SpecimenConc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278.15 K / Details: blot time = 7s blot force = 4

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 69 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.15particle selection
2SerialEM3.8.6image acquisition
3DigitalMicrograph3.31.2359.0image acquisition
5cryoSPARC2.15CTF correction
10Rosetta3model refinement
11cryoSPARC2.15initial Euler assignment
13RELION3.1.0classification
14RELION3.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32734 / Symmetry type: POINT

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