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- PDB-6pp9: Crystal structure of BRAF:MEK1 complex -

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Basic information

Entry
Database: PDB / ID: 6pp9
TitleCrystal structure of BRAF:MEK1 complex
Components
  • Dual specificity mitogen-activated protein kinase kinase 1
  • Serine/threonine-protein kinase B-raf
KeywordsTRANSFERASE / BRAF / MEK1
Function / homology
Function and homology information


negative regulation of homotypic cell-cell adhesion / regulation of vascular associated smooth muscle contraction / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / mitogen-activated protein kinase kinase / melanosome transport / Golgi inheritance / Signalling to p38 via RIT and RIN / MAP kinase scaffold activity / positive regulation of muscle contraction / ARMS-mediated activation ...negative regulation of homotypic cell-cell adhesion / regulation of vascular associated smooth muscle contraction / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / mitogen-activated protein kinase kinase / melanosome transport / Golgi inheritance / Signalling to p38 via RIT and RIN / MAP kinase scaffold activity / positive regulation of muscle contraction / ARMS-mediated activation / establishment of protein localization to membrane / Signaling by MAP2K mutants / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / positive regulation of D-glucose transmembrane transport / vesicle transport along microtubule / regulation of Golgi inheritance / mitogen-activated protein kinase kinase kinase binding / positive regulation of protein serine/threonine kinase activity / triglyceride homeostasis / regulation of early endosome to late endosome transport / Negative feedback regulation of MAPK pathway / regulation of stress-activated MAPK cascade / Frs2-mediated activation / MAPK3 (ERK1) activation / ERBB2-ERBB3 signaling pathway / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / MAP kinase kinase activity / response to axon injury / positive regulation of ATP biosynthetic process / neuromuscular junction development / ERK1 and ERK2 cascade / Uptake and function of anthrax toxins / positive regulation of peptidyl-serine phosphorylation / MAP kinase kinase kinase activity / protein kinase activator activity / Schwann cell development / postsynaptic modulation of chemical synaptic transmission / myelination / insulin-like growth factor receptor signaling pathway / animal organ morphogenesis / protein serine/threonine/tyrosine kinase activity / cellular response to calcium ion / positive regulation of autophagy / neuron projection morphogenesis / response to glucocorticoid / protein serine/threonine kinase activator activity / dendrite cytoplasm / Signal transduction by L1 / MAP3K8 (TPL2)-dependent MAPK1/3 activation / positive regulation of transcription elongation by RNA polymerase II / RAF activation / Signaling by high-kinase activity BRAF mutants / Spry regulation of FGF signaling / MAP2K and MAPK activation / cellular senescence / chemotaxis / small GTPase binding / epidermal growth factor receptor signaling 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 / MAPK cascade / Negative regulation of MAPK pathway / Signaling by BRAF and RAF1 fusions / neuron differentiation / late endosome / ciliary basal body / response to oxidative stress / protein tyrosine kinase activity / cell body / scaffold protein binding / microtubule / cell cortex / early endosome / perikaryon / positive regulation of ERK1 and ERK2 cascade / protein kinase activity / protein phosphorylation / non-specific serine/threonine protein kinase / postsynapse / neuron projection / postsynaptic density / positive regulation of cell migration / negative regulation of cell population proliferation / negative regulation of gene expression / axon / protein serine kinase activity / focal adhesion / protein serine/threonine kinase activity / centrosome / positive regulation of gene expression / calcium ion binding / negative regulation of apoptotic process / positive regulation of DNA-templated transcription / protein-containing complex binding / perinuclear region of cytoplasm / glutamatergic synapse / Golgi apparatus
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. ...: / 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 / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Ubiquitin-like domain superfamily / Phosphorylase Kinase; domain 1 / Phosphorylase Kinase; domain 1 / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / 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 / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Chem-LCJ / Serine/threonine-protein kinase B-raf / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.59 Å
AuthorsLi, K. / Gonzalez Del-Pino, G. / Park, E. / Eck, M.J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)P50 CA165962 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R50 CA221830 United States
CitationJournal: Nature / Year: 2019
Title: Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes.
Authors: Eunyoung Park / Shaun Rawson / Kunhua Li / Byeong-Won Kim / Scott B Ficarro / Gonzalo Gonzalez-Del Pino / Humayun Sharif / Jarrod A Marto / Hyesung Jeon / Michael J Eck /
Abstract: RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival. RAF activity is tightly ...RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.
History
DepositionJul 5, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Dec 4, 2019Group: Author supporting evidence / Database references / Category: citation / pdbx_audit_support
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _pdbx_audit_support.funding_organization
Revision 1.3Oct 11, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Serine/threonine-protein kinase B-raf
B: Dual specificity mitogen-activated protein kinase kinase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)77,75712
Polymers75,8882
Non-polymers1,86810
Water1,29772
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5570 Å2
ΔGint-61 kcal/mol
Surface area25550 Å2
MethodPISA
Unit cell
Length a, b, c (Å)116.697, 116.697, 130.944
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121

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Components

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Protein , 2 types, 2 molecules AB

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


Mass: 32098.104 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRAF, BRAF1, RAFB1 / Plasmid: pFASTBac Dual / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P15056, non-specific serine/threonine protein kinase
#2: Protein Dual specificity mitogen-activated protein kinase kinase 1 / MKK1 / ERK activator kinase 1 / MAPK/ERK kinase 1 / MEK 1


Mass: 43790.281 Da / Num. of mol.: 1 / Mutation: S218A, S222A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP2K1, MEK1, PRKMK1 / Plasmid: PAC8 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q02750, mitogen-activated protein kinase kinase

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Non-polymers , 7 types, 82 molecules

#3: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-LCJ / 5-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)imidazo[1,5-a]pyridine-6-carboxamide


Mass: 456.210 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H14FIN4O3 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#8: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 72 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.39 Å3/Da / Density % sol: 67.29 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 6.4
Details: 100 mM Bis-Tris pH 6.4, 200 mM Ammonium Sulfate, and 22% PEG3350
PH range: 6.0-6.8 / Temp details: RT

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.978 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 11, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978 Å / Relative weight: 1
ReflectionResolution: 2.58→43.648 Å / Num. obs: 32549 / % possible obs: 99.95 % / Redundancy: 13.3 % / Biso Wilson estimate: 52.17 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.215 / Rpim(I) all: 0.06127 / Rrim(I) all: 0.2237 / Net I/σ(I): 10.86
Reflection shellResolution: 2.58→2.683 Å / Redundancy: 13.9 % / Mean I/σ(I) obs: 2 / Num. unique obs: 3190 / CC1/2: 0.888 / Rpim(I) all: 0.4253 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.15.2_3472refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6NYB

6nyb


Resolution: 2.59→43.648 Å / SU ML: 0.31 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.7
RfactorNum. reflection% reflection
Rfree0.2455 1543 4.74 %
Rwork0.2207 --
obs0.2218 32544 99.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 122.96 Å2 / Biso mean: 53.5009 Å2 / Biso min: 27.5 Å2
Refinement stepCycle: final / Resolution: 2.59→43.648 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4667 0 108 72 4847
Biso mean--48.04 48.47 -
Num. residues----590
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork
2.5901-2.67370.35581330.27132779
2.6737-2.76930.31171630.26252763
2.7693-2.88010.30211350.25142761
2.8801-3.01120.28031210.24332820
3.0112-3.16990.27491640.25382765
3.1699-3.36840.26131250.22922816
3.3684-3.62840.24291380.21052804
3.6284-3.99330.2191370.20092821
3.9933-4.57060.21241560.18262809
4.5706-5.75640.22081340.20372876
5.7564-43.6480.24281370.24352987

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