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- PDB-8vys: Cryo-EM Structure of the BRAF V600E monomer bound to PLX8394 -

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Basic information

Entry
Database: PDB / ID: 8vys
TitleCryo-EM Structure of the BRAF V600E monomer bound to PLX8394
Components
  • 14-3-3 protein zeta/delta
  • Serine/threonine-protein kinase B-raf
KeywordsTRANSFERASE/INHIBITOR / BRAF Kinase Monomer Mutant Inhibitor / TRANSFERASE-INHIBITOR complex
Function / homology
Function and homology information


synaptic target recognition / Golgi reassembly / CD4-positive, alpha-beta T cell differentiation / NOTCH4 Activation and Transmission of Signal to the Nucleus / positive regulation of axon regeneration / myeloid progenitor cell differentiation / respiratory system process / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / establishment of Golgi localization ...synaptic target recognition / Golgi reassembly / CD4-positive, alpha-beta T cell differentiation / NOTCH4 Activation and Transmission of Signal to the Nucleus / positive regulation of axon regeneration / myeloid progenitor cell differentiation / respiratory system process / CD4-positive or CD8-positive, alpha-beta T cell lineage commitment / negative regulation of synaptic vesicle exocytosis / establishment of Golgi localization / tube formation / Signalling to p38 via RIT and RIN / head morphogenesis / endothelial cell apoptotic process / ARMS-mediated activation / negative regulation of fibroblast migration / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / regulation of synapse maturation / Rap1 signalling / positive regulation of D-glucose transmembrane transport / establishment of protein localization to membrane / positive regulation of axonogenesis / regulation of T cell differentiation / negative regulation of protein localization to nucleus / face development / KSRP (KHSRP) binds and destabilizes mRNA / somatic stem cell population maintenance / thyroid gland development / Negative feedback regulation of MAPK pathway / GP1b-IX-V activation signalling / Frs2-mediated activation / stress fiber assembly / MAP kinase kinase activity / Regulation of localization of FOXO transcription factors / Interleukin-3, Interleukin-5 and GM-CSF signaling / lung development / synaptic vesicle exocytosis / positive regulation of peptidyl-serine phosphorylation / phosphoserine residue binding / Activation of BAD and translocation to mitochondria / negative regulation of endothelial cell apoptotic process / MAP kinase kinase kinase activity / ERK1 and ERK2 cascade / centriolar satellite / regulation of ERK1 and ERK2 cascade / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / thymus development / postsynaptic modulation of chemical synaptic transmission / cellular response to glucose starvation / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / positive regulation of stress fiber assembly / RHO GTPases activate PKNs / substrate adhesion-dependent cell spreading / positive regulation of substrate adhesion-dependent cell spreading / negative regulation of TORC1 signaling / Transcriptional and post-translational regulation of MITF-M expression and activity / animal organ morphogenesis / cellular response to calcium ion / negative regulation of innate immune response / hippocampal mossy fiber to CA3 synapse / TP53 Regulates Metabolic Genes / Translocation of SLC2A4 (GLUT4) to the plasma membrane / sperm end piece / sperm principal piece / cellular response to xenobiotic stimulus / regulation of protein stability / protein sequestering activity / Deactivation of the beta-catenin transactivating complex / visual learning / RAF activation / Signaling by high-kinase activity BRAF mutants / Spry regulation of FGF signaling / MAP2K and MAPK activation / Negative regulation of NOTCH4 signaling / long-term synaptic potentiation / epidermal growth factor receptor signaling pathway / T cell differentiation in thymus / intracellular protein localization / 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 / MAPK cascade / Negative regulation of MAPK pathway / melanosome / Signaling by BRAF and RAF1 fusions / regulation of cell population proliferation / T cell receptor signaling pathway / sperm midpiece / presynapse / angiogenesis / cell body / scaffold protein binding / cilium / protein phosphatase binding / blood microparticle / negative regulation of neuron apoptotic process / DNA-binding transcription factor binding
Similarity search - Function
Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Phorbol esters/diacylglycerol binding domain (C1 domain) / : / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) ...Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / Phorbol esters/diacylglycerol binding domain (C1 domain) / : / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / C1-like domain superfamily / 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 / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Ubiquitin-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
: / Serine/threonine-protein kinase B-raf / 14-3-3 protein zeta/delta
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsLavoie, H. / Lajoie, D. / Jin, T. / Decossas, M. / Maisonneuve, P. / Therrien, M.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FDN-388023 Canada
CitationJournal: Science / Year: 2025
Title: BRAF oncogenic mutants evade autoinhibition through a common mechanism.
Authors: Hugo Lavoie / Ting Jin / Driss Lajoie / Marion Decossas / Patrick Gendron / Bing Wang / Frantisek Filandr / Malha Sahmi / Chang Hwa Jo / Sandra Weber / Geneviève Arseneault / Sasmita ...Authors: Hugo Lavoie / Ting Jin / Driss Lajoie / Marion Decossas / Patrick Gendron / Bing Wang / Frantisek Filandr / Malha Sahmi / Chang Hwa Jo / Sandra Weber / Geneviève Arseneault / Sasmita Tripathy / Pierre Beaulieu / Doris A Schuetz / David C Schriemer / Anne Marinier / William J Rice / Pierre Maisonneuve / Marc Therrien /
Abstract: Uncontrolled activation of the rat sarcoma (RAS)-extracellular signal-regulated kinase (ERK) pathway drives tumor growth, often because of oncogenic BRAF mutations. BRAF regulation, involving ...Uncontrolled activation of the rat sarcoma (RAS)-extracellular signal-regulated kinase (ERK) pathway drives tumor growth, often because of oncogenic BRAF mutations. BRAF regulation, involving monomeric autoinhibition and activation by dimerization, has been intensely scrutinized, but mechanisms enabling oncogenic mutants to evade regulation remain unclear. By using cryo-electron microscopy, we solved the three-dimensional structures of the three oncogenic BRAF mutant classes, including the common V600E variant. These mutations disrupted wild-type BRAF's autoinhibited state, mediated by interactions between the cysteine-rich domain and kinase domain, thereby shifting the kinase domain into a preactivated conformation. This structural change likely results from helix αC displacement. PLX8394, a BRAF inhibitor that stabilizes helix αC in an inactive conformation, restored the autoinhibited conformation of oncogenic BRAF, explaining the properties of this class of compounds.
History
DepositionFeb 9, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 28, 2025Provider: repository / Type: Initial release
Revision 1.1Jun 11, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Jun 25, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Serine/threonine-protein kinase B-raf
B: 14-3-3 protein zeta/delta
C: 14-3-3 protein zeta/delta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,6354
Polymers140,0933
Non-polymers5431
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Serine/threonine-protein kinase B-raf / Proto-oncogene B-Raf / p94 / v-Raf murine sarcoma viral oncogene homolog B1


Mass: 84800.750 Da / Num. of mol.: 1 / Mutation: V600E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell: FreeStyle 293-F cells / Gene: BRAF, BRAF1, RAFB1 / Plasmid: pCDNA3.1-FLAG-TEV-BRAF V600E / Cell (production host): FreeStyle 293-F cells / Production host: Homo sapiens (human)
References: UniProt: P15056, non-specific serine/threonine protein kinase
#2: Protein 14-3-3 protein zeta/delta / Protein kinase C inhibitor protein 1 / KCIP-1


Mass: 27645.895 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: FreeStyle 293-F cells / References: UniProt: P63104
#3: Chemical ChemComp-A1AEN / (3S)-N-{3-[5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl}-3-fluoropyrrolidine-1-sulfonamide


Mass: 542.533 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H21F3N6O3S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: BRAF V600E - 14.3.3 complex bound to PLX8394 / Type: COMPLEX
Details: BRAF complex purified by Flag affinity purification followed by TEV elution from FreeStyle 293F cells
Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.141 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: FreeStyle 293-F cells / Plasmid: pCDNA3.1-FLAG-TEV-BRAF V600E
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H17N2NaO4S1
2137 mMSodium ChlorideNaCl1
35 mMMagnesium chlorideMgCl21
41 mMTCEPC9H15O6P1
50.0005 mMATPgammaSC10H12Li4N5O12P3S1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 80 % / Details: Chameleon system (SPT Labtech)

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 56.44 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4CTFFIND4CTF correction
8PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177000
Details: Resolution corresponds to the FSC 0.143 cut-off of the masked map.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036314
ELECTRON MICROSCOPYf_angle_d0.4548501
ELECTRON MICROSCOPYf_dihedral_angle_d11.0252424
ELECTRON MICROSCOPYf_chiral_restr0.034930
ELECTRON MICROSCOPYf_plane_restr0.0031094

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