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- PDB-8dp5: Structure of the PEAK3/14-3-3 complex -

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Basic information

Entry
Database: PDB / ID: 8dp5
TitleStructure of the PEAK3/14-3-3 complex
Components
  • 14-3-3 protein beta/alpha
  • 14-3-3 protein epsilon
  • Protein PEAK3
  • Protein PEAK3 fragment
KeywordsSIGNALING PROTEIN / complex / pseudokinase / kinase / adapter
Function / homology
Function and homology information


negative regulation of peptidyl-serine dephosphorylation / regulation of heart rate by hormone / negative regulation of protein dephosphorylation / regulation of potassium ion transmembrane transporter activity / negative regulation of calcium ion transmembrane transporter activity / membrane repolarization during cardiac muscle cell action potential / cytoplasmic sequestering of protein / Tristetraprolin (TTP, ZFP36) binds and destabilizes mRNA / negative regulation of G protein-coupled receptor signaling pathway / Butyrate Response Factor 1 (BRF1) binds and destabilizes mRNA ...negative regulation of peptidyl-serine dephosphorylation / regulation of heart rate by hormone / negative regulation of protein dephosphorylation / regulation of potassium ion transmembrane transporter activity / negative regulation of calcium ion transmembrane transporter activity / membrane repolarization during cardiac muscle cell action potential / cytoplasmic sequestering of protein / Tristetraprolin (TTP, ZFP36) binds and destabilizes mRNA / negative regulation of G protein-coupled receptor signaling pathway / Butyrate Response Factor 1 (BRF1) binds and destabilizes mRNA / regulation of membrane repolarization / MTOR signalling / NADE modulates death signalling / RAB GEFs exchange GTP for GDP on RABs / ARMS-mediated activation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / Signaling by Hippo / vacuolar membrane / negative regulation of calcium ion export across plasma membrane / Frs2-mediated activation / Deregulated CDK5 triggers multiple neurodegenerative pathways in Alzheimer's disease models / protein kinase inhibitor activity / positive regulation of catalytic activity / cytoplasmic pattern recognition receptor signaling pathway / mTORC1-mediated signalling / regulation of heart rate by cardiac conduction / calcium channel regulator activity / Regulation of localization of FOXO transcription factors / protein localization to nucleus / phosphoserine residue binding / HSF1 activation / protein targeting / Regulation of HSF1-mediated heat shock response / Activation of BAD and translocation to mitochondria / potassium channel regulator activity / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / signaling adaptor activity / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / RHO GTPases activate PKNs / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / Recruitment of NuMA to mitotic centrosomes / regulation of mitotic cell cycle / regulation of cytosolic calcium ion concentration / Anchoring of the basal body to the plasma membrane / substantia nigra development / AURKA Activation by TPX2 / protein sequestering activity / positive regulation of protein export from nucleus / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of actin cytoskeleton organization / mitochondrial membrane / hippocampus development / TP53 Regulates Metabolic Genes / phosphoprotein binding / RAF activation / neuron migration / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / cerebral cortex development / Negative regulation of MAPK pathway / histone deacetylase binding / 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 / : / Regulation of PLK1 Activity at G2/M Transition / MAPK cascade / Signaling by BRAF and RAF1 fusions / melanosome / actin cytoskeleton / MHC class II protein complex binding / cellular response to heat / regulation of cell shape / scaffold protein binding / protein phosphatase binding / transmembrane transporter binding / protein kinase activity / intracellular signal transduction / cadherin binding / protein heterodimerization activity / protein domain specific binding / protein phosphorylation / focal adhesion / ubiquitin protein ligase binding / perinuclear region of cytoplasm / enzyme binding / signal transduction / RNA binding / extracellular exosome / membrane / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
14-3-3 proteins signature 2. / 14-3-3 protein, conserved site / 14-3-3 proteins signature 1. / 14-3-3 protein / 14-3-3 homologues / 14-3-3 domain / 14-3-3 domain superfamily / 14-3-3 protein
Similarity search - Domain/homology
14-3-3 protein beta/alpha / 14-3-3 protein epsilon / Protein PEAK3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 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 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein PEAK3
B: Protein PEAK3
C: 14-3-3 protein beta/alpha
D: 14-3-3 protein epsilon
E: Protein PEAK3 fragment
P: Protein PEAK3 fragment


Theoretical massNumber of molelcules
Total (without water)266,9116
Polymers266,9116
Non-polymers00
Water0
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 / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: Q6ZS72
#2: Protein 14-3-3 protein beta/alpha / Protein 1054 / Protein kinase C inhibitor protein 1 / KCIP-1


Mass: 28114.373 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: YWHAB / Production host: Homo sapiens (human) / References: UniProt: P31946
#3: Protein 14-3-3 protein epsilon / 14-3-3E


Mass: 29208.900 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: YWHAE / Production host: Homo sapiens (human) / References: UniProt: P62258
#4: Protein Protein PEAK3 fragment


Mass: 52437.012 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
Compound detailsThe authors state that chains E and P are part of Chains A and B. However, because they cannot ...The authors state that chains E and P are part of Chains A and B. However, because they cannot resolve a large portion of the N-terminal segments of Chain A and B and therefore the connectivity between these two sets of chains, they cannot with confidence assign Chain E residues to Chains A or B and the same with Chain P residues.
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: Complex between PEAK3 and 14-3-3 epsilon, beta / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.1618 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 ChlorideNaClSodium chloride1
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 FIELDBright-field microscopy / 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
10cryoSPARC2.15initial Euler assignment
12cryoSPARC2.15classification
13cryoSPARC2.153D reconstruction
14Coot0.9.6model refinement
15ISOLDE1.2.1model refinement
16Rosetta3model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169563 / Symmetry type: POINT

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