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- EMDB-27630: Structure of the PEAK3/14-3-3 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-27630
TitleStructure of the PEAK3/14-3-3 complex
Map dataReconstruction of the PEAK3/14-3-3 complex filtered to 3.1A and sharpened with 107.4 bFactor.
Sample
  • Complex: Complex between PEAK3 and 14-3-3 epsilon, beta
    • Protein or peptide: Protein PEAK3
    • Protein or peptide: 14-3-3 protein beta/alpha
    • Protein or peptide: 14-3-3 protein epsilon
    • Protein or peptide: Protein PEAK3 fragment
Keywordscomplex / pseudokinase / kinase / adapter / SIGNALING PROTEIN
Function / homology
Function and homology information


negative regulation of peptidyl-serine dephosphorylation / regulation of heart rate by hormone / regulation of potassium ion transmembrane transporter activity / negative regulation of calcium ion transmembrane transporter activity / negative regulation of protein dephosphorylation / membrane repolarization during cardiac muscle cell action potential / cytoplasmic sequestering of protein / Tristetraprolin (TTP, ZFP36) binds and destabilizes mRNA / negative regulation of toll-like receptor signaling pathway / negative regulation of G protein-coupled receptor signaling pathway ...negative regulation of peptidyl-serine dephosphorylation / regulation of heart rate by hormone / regulation of potassium ion transmembrane transporter activity / negative regulation of calcium ion transmembrane transporter activity / negative regulation of protein dephosphorylation / membrane repolarization during cardiac muscle cell action potential / cytoplasmic sequestering of protein / Tristetraprolin (TTP, ZFP36) binds and destabilizes mRNA / negative regulation of toll-like receptor signaling pathway / negative regulation of G protein-coupled receptor signaling pathway / Butyrate Response Factor 1 (BRF1) binds and destabilizes mRNA / regulation of membrane repolarization / protein localization to endoplasmic reticulum / 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 / cytoplasmic pattern recognition receptor signaling pathway / Frs2-mediated activation / Deregulated CDK5 triggers multiple neurodegenerative pathways in Alzheimer's disease models / protein kinase inhibitor activity / positive regulation of catalytic activity / mTORC1-mediated signalling / regulation of heart rate by cardiac conduction / protein localization to nucleus / Regulation of localization of FOXO transcription factors / calcium channel regulator activity / phosphoserine residue binding / Regulation of HSF1-mediated heat shock response / Activation of BAD and translocation to mitochondria / HSF1 activation / potassium channel regulator activity / protein targeting / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / signaling adaptor activity / 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 / Anchoring of the basal body to the plasma membrane / protein sequestering activity / regulation of mitotic cell cycle / regulation of cytosolic calcium ion concentration / substantia nigra development / AURKA Activation by TPX2 / positive regulation of protein export from nucleus / Translocation of SLC2A4 (GLUT4) to the plasma membrane / mitochondrial membrane / hippocampus development / regulation of actin cytoskeleton organization / phosphoprotein binding / TP53 Regulates Metabolic Genes / RAF activation / neuron migration / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / cerebral cortex development / histone deacetylase binding / Negative regulation of MAPK pathway / 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 / focal adhesion / ubiquitin protein ligase binding / perinuclear region of cytoplasm / enzyme binding / endoplasmic reticulum / signal transduction / RNA binding / extracellular exosome / membrane / identical protein binding / nucleus
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsTorosyan H / Paul M / Jura N / Verba KA
Funding support United States, 3 items
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, 2022-
Header (metadata) releaseJun 28, 2023-
Map releaseJun 28, 2023-
UpdateJun 28, 2023-
Current statusJun 28, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27630.png

Downloads & links

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Map

FileDownload / File: emd_27630.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the PEAK3/14-3-3 complex filtered to 3.1A and sharpened with 107.4 bFactor.
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.268
Minimum - Maximum-1.2761475 - 2.3827164
Average (Standard dev.)0.00044206422 (±0.05109717)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 267.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27630_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered, unsharpened half map.

Fileemd_27630_half_map_1.map
AnnotationUnfiltered, unsharpened half map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered, unsharpened half map.

Fileemd_27630_half_map_2.map
AnnotationUnfiltered, unsharpened half map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex between PEAK3 and 14-3-3 epsilon, beta

EntireName: Complex between PEAK3 and 14-3-3 epsilon, beta
Components
  • Complex: Complex between PEAK3 and 14-3-3 epsilon, beta
    • Protein or peptide: Protein PEAK3
    • Protein or peptide: 14-3-3 protein beta/alpha
    • Protein or peptide: 14-3-3 protein epsilon
    • Protein or peptide: Protein PEAK3 fragment

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Supramolecule #1: Complex between PEAK3 and 14-3-3 epsilon, beta

SupramoleculeName: Complex between PEAK3 and 14-3-3 epsilon, beta / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 161.8 KDa

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Macromolecule #1: Protein PEAK3

MacromoleculeName: Protein PEAK3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.357031 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSSPEPPTEP PEPDNPTWST QPTYSNLGQI RAHLLPSKAC RLRTPGSLST NPEPLPPPLP KKILTRTQSL PTRRTLHPSS IQVQPPRRP FLGSHSVDKS QAAVGPACLP AELTFGPADA PLGLSLRDLH SPEAVHTALA ARQLQGLRTI YARLRARLMG G HPGPCHPG ...String:
MSSPEPPTEP PEPDNPTWST QPTYSNLGQI RAHLLPSKAC RLRTPGSLST NPEPLPPPLP KKILTRTQSL PTRRTLHPSS IQVQPPRRP FLGSHSVDKS QAAVGPACLP AELTFGPADA PLGLSLRDLH SPEAVHTALA ARQLQGLRTI YARLRARLMG G HPGPCHPG HSFRLLDSSP CAESGDALYY RVVRAHEDAW HILVAKVPKP GADVPHPWGL ELQASLSPHF NLQGLCGLVP EG TLPGAPW RGAVALAAEV PERTVAQWLA EACTQPPEEF VWAVALLLLQ LSAALKFLEA WGAALVELRP ENLLLVAPRG CAT TGPPRL LLTDFGRVCL QPPGPPGSPG PHAPQLGSLL RALLSLAAPS TTPLAAGLEL LAAQLTRLRP SASRTRGALQ ALLW GPGPE LRGRGAPLGP WLRALGPWLR VRRGLLVLRL AERAAGGEAP SLEDWLCCEY LAEATESSMG QALALLWDLE GGGGA DYKD DDDKGPV

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Macromolecule #2: 14-3-3 protein beta/alpha

MacromoleculeName: 14-3-3 protein beta/alpha / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.114373 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTMDKSELVQ KAKLAEQAER YDDMAAAMKA VTEQGHELSN EERNLLSVAY KNVVGARRSS WRVISSIEQK TERNEKKQQM GKEYREKIE AELQDICNDV LELLDKYLIP NATQPESKVF YLKMKGDYFR YLSEVASGDN KQTTVSNSQQ AYQEAFEISK K EMQPTHPI ...String:
MTMDKSELVQ KAKLAEQAER YDDMAAAMKA VTEQGHELSN EERNLLSVAY KNVVGARRSS WRVISSIEQK TERNEKKQQM GKEYREKIE AELQDICNDV LELLDKYLIP NATQPESKVF YLKMKGDYFR YLSEVASGDN KQTTVSNSQQ AYQEAFEISK K EMQPTHPI RLGLALNFSV FYYEILNSPE KACSLAKTAF DEAIAELDTL NEESYKDSTL IMQLLRDNLT LWTSENQGDE GD AGEGEN

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Macromolecule #3: 14-3-3 protein epsilon

MacromoleculeName: 14-3-3 protein epsilon / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 29.2089 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDDREDLVYQ AKLAEQAERY DEMVESMKKV AGMDVELTVE ERNLLSVAYK NVIGARRASW RIISSIEQKE ENKGGEDKLK MIREYRQMV ETELKLICCD ILDVLDKHLI PAANTGESKV FYYKMKGDYH RYLAEFATGN DRKEAAENSL VAYKAASDIA M TELPPTHP ...String:
MDDREDLVYQ AKLAEQAERY DEMVESMKKV AGMDVELTVE ERNLLSVAYK NVIGARRASW RIISSIEQKE ENKGGEDKLK MIREYRQMV ETELKLICCD ILDVLDKHLI PAANTGESKV FYYKMKGDYH RYLAEFATGN DRKEAAENSL VAYKAASDIA M TELPPTHP IRLGLALNFS VFYYEILNSP DRACRLAKAA FDDAIAELDT LSEESYKDST LIMQLLRDNL TLWTSDMQGD GE EQNKEAL QDVEDENQ

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Macromolecule #4: Protein PEAK3 fragment

MacromoleculeName: Protein PEAK3 fragment / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.437012 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSSPEPPTEP PEPDNPTWST QPTYSNLGQI RAHLLPSKAC RLRTPGSLST NPEPLPPPLP KKILTRTQ(SEP)L PTRRTL HPS SIQVQPPRRP FLGSHSVDKS QAAVGPACLP AELTFGPADA PLGLSLRDLH SPEAVHTALA ARQLQGLRTI YARLRAR LM GGHPGPCHPG ...String:
MSSPEPPTEP PEPDNPTWST QPTYSNLGQI RAHLLPSKAC RLRTPGSLST NPEPLPPPLP KKILTRTQ(SEP)L PTRRTL HPS SIQVQPPRRP FLGSHSVDKS QAAVGPACLP AELTFGPADA PLGLSLRDLH SPEAVHTALA ARQLQGLRTI YARLRAR LM GGHPGPCHPG HSFRLLDSSP CAESGDALYY RVVRAHEDAW HILVAKVPKP GADVPHPWGL ELQASLSPHF NLQGLCGL V PEGTLPGAPW RGAVALAAEV PERTVAQWLA EACTQPPEEF VWAVALLLLQ LSAALKFLEA WGAALVELRP ENLLLVAPR GCATTGPPRL LLTDFGRVCL QPPGPPGSPG PHAPQLGSLL RALLSLAAPS TTPLAAGLEL LAAQLTRLRP SASRTRGALQ ALLWGPGPE LRGRGAPLGP WLRALGPWLR VRRGLLVLRL AERAAGGEAP SLEDWLCCEY LAEATESSMG QALALLWDLE G GGGADYKD DDDKGPV

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMNH2C(CH2OH)3Tris Base
150.0 mMNaClSodium Chloride
2.0 mMC4H10O2S2Dithiothreitol
2.0 mMMgCl2Magnesium Chloride

Details: A final concentration of 0.1% of Octyl-beta-Glucoside (C14H28O6) was added to the sample before freezing.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot time = 7s blot force = 4.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 69.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 169563
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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