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- PDB-7mfd: Autoinhibited BRAF:(14-3-3)2:MEK complex with the BRAF RBD resolved -

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Basic information

Entry
Database: PDB / ID: 7mfd
TitleAutoinhibited BRAF:(14-3-3)2:MEK complex with the BRAF RBD resolved
Components
  • 14-3-3 protein zeta/delta
  • Dual specificity mitogen-activated protein kinase kinase 1
  • Serine/threonine-protein kinase B-raf
KeywordsSIGNALING PROTEIN/TRANSFERASE / B-Raf / MEK / 14-3-3 / B-Raf complex / B-Raf monomer / Inactive B-Raf / Serine/threonine-protein kinase B-raf / RBD / signaling protein / SIGNALING PROTEIN-TRANSFERASE complex
Function / homology
Function and homology information


synaptic target recognition / negative regulation of homotypic cell-cell adhesion / Golgi reassembly / regulation of vascular associated smooth muscle contraction / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / NOTCH4 Activation and Transmission of Signal to the Nucleus / respiratory system process / mitogen-activated protein kinase kinase / establishment of Golgi localization / melanosome transport ...synaptic target recognition / negative regulation of homotypic cell-cell adhesion / Golgi reassembly / regulation of vascular associated smooth muscle contraction / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / NOTCH4 Activation and Transmission of Signal to the Nucleus / respiratory system process / mitogen-activated protein kinase kinase / establishment of Golgi localization / melanosome transport / tube formation / Golgi inheritance / MAP kinase scaffold activity / Signalling to p38 via RIT and RIN / positive regulation of muscle contraction / ARMS-mediated activation / Signaling by MAP2K mutants / regulation of synapse maturation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / positive regulation of D-glucose transmembrane transport / establishment of protein localization to membrane / vesicle transport along microtubule / regulation of Golgi inheritance / mitogen-activated protein kinase kinase kinase binding / negative regulation of protein localization to nucleus / KSRP (KHSRP) binds and destabilizes mRNA / triglyceride homeostasis / positive regulation of protein serine/threonine kinase activity / regulation of early endosome to late endosome transport / Negative feedback regulation of MAPK pathway / regulation of stress-activated MAPK cascade / GP1b-IX-V activation signalling / Frs2-mediated activation / MAPK3 (ERK1) activation / ERBB2-ERBB3 signaling pathway / MAP kinase kinase activity / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / lung development / positive regulation of ATP biosynthetic process / neuromuscular junction development / ERK1 and ERK2 cascade / response to axon injury / Regulation of localization of FOXO transcription factors / Interleukin-3, Interleukin-5 and GM-CSF signaling / Uptake and function of anthrax toxins / positive regulation of peptidyl-serine phosphorylation / phosphoserine residue binding / Activation of BAD and translocation to mitochondria / regulation of ERK1 and ERK2 cascade / MAP kinase kinase kinase activity / protein kinase activator activity / Schwann cell development / postsynaptic modulation of chemical synaptic transmission / cellular response to glucose starvation / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / RHO GTPases activate PKNs / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / myelination / insulin-like growth factor receptor signaling pathway / negative regulation of TORC1 signaling / animal organ morphogenesis / protein serine/threonine/tyrosine kinase activity / Transcriptional and post-translational regulation of MITF-M expression and activity / cellular response to calcium ion / neuron projection morphogenesis / response to glucocorticoid / negative regulation of innate immune response / positive regulation of autophagy / protein serine/threonine kinase activator activity / dendrite cytoplasm / hippocampal mossy fiber to CA3 synapse / Signal transduction by L1 / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TP53 Regulates Metabolic Genes / Translocation of SLC2A4 (GLUT4) to the plasma membrane / positive regulation of transcription elongation by RNA polymerase II / regulation of protein stability / RAF activation / protein sequestering activity / Signaling by high-kinase activity BRAF mutants / Spry regulation of FGF signaling / Deactivation of the beta-catenin transactivating complex / MAP2K and MAPK activation / cellular senescence / Negative regulation of NOTCH4 signaling / epidermal growth factor receptor signaling pathway / chemotaxis / intracellular protein localization / small GTPase 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 / MAPK cascade / Negative regulation of MAPK pathway / Signaling by BRAF and RAF1 fusions / melanosome
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 / 14-3-3 domain / Delta-Endotoxin; domain 1 / Phorbol esters/diacylglycerol binding domain (C1 domain) / : ...: / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / 14-3-3 domain / Delta-Endotoxin; domain 1 / 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. / Protein kinase domain / 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 / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Chem-CHU / Serine/threonine-protein kinase B-raf / 14-3-3 protein zeta/delta / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsMartinez Fiesco, J.A. / Ping, Z. / Durrant, D.E. / Morrison, D.K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC 011744 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC 010329 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding.
Authors: Juliana A Martinez Fiesco / David E Durrant / Deborah K Morrison / Ping Zhang /
Abstract: RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present ...RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-3:MEK and BRAF:14-3-3 complexes, and an inhibitor-bound, dimeric BRAF:14-3-3 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminal site and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.
History
DepositionApr 9, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
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Revision 1.2Oct 16, 2024Group: Data collection / Structure summary
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Assembly

Deposited unit
A: Serine/threonine-protein kinase B-raf
B: Dual specificity mitogen-activated protein kinase kinase 1
C: 14-3-3 protein zeta/delta
D: 14-3-3 protein zeta/delta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)184,3487
Polymers183,7464
Non-polymers6023
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
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: 84697.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRAF, BRAF1, RAFB1 / Production host: Homo sapiens (human)
References: UniProt: P15056, non-specific serine/threonine protein kinase
#2: Protein Dual specificity mitogen-activated protein kinase kinase 1 / MAP kinase kinase 1 / MAPKK 1 / MKK1 / ERK activator kinase 1 / MAPK/ERK kinase 1 / MEK 1


Mass: 43493.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
References: UniProt: Q02750, mitogen-activated protein kinase kinase
#3: Protein 14-3-3 protein zeta/delta / Protein kinase C inhibitor protein 1 / KCIP-1


Mass: 27777.092 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P63104
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-CHU / N-(3-fluoro-4-{[4-methyl-2-oxo-7-(pyrimidin-2-yloxy)-2H-chromen-3-yl]methyl}pyridin-2-yl)-N'-methylsulfuric diamide / CH5126766


Mass: 471.462 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H18FN5O5S
Has ligand of interestN
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

Component
IDNameTypeEntity IDParent-IDSource
1Autoinhibited B-Raf:(14-3-3)2:MEK complex with resolved RBDCOMPLEX#1-#30MULTIPLE SOURCES
2B-rafCOMPLEX#11RECOMBINANT
3MEKCOMPLEX#21NATURAL
414-3-3 protein zeta/deltaCOMPLEX#31NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2200 mMSodium chlorideNaCl1
310 mMDTT1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20mAmp / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K

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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
Image recordingElectron dose: 57 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142852 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049293
ELECTRON MICROSCOPYf_angle_d0.66512529
ELECTRON MICROSCOPYf_dihedral_angle_d15.3273512
ELECTRON MICROSCOPYf_chiral_restr0.0451386
ELECTRON MICROSCOPYf_plane_restr0.0051631

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