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- PDB-7z37: Structure of the RAF1-HSP90-CDC37 complex (RHC-II) -

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Basic information

Entry
Database: PDB / ID: 7z37
TitleStructure of the RAF1-HSP90-CDC37 complex (RHC-II)
Components
  • Heat shock protein HSP 90-beta
  • Hsp90 co-chaperone Cdc37
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsTRANSFERASE / RAF1-HSP90-CDC37 / complex / proto-oncogene / serine/threonine kinase / cancer / chaperone
Function / homology
Function and homology information


regulation of type II interferon-mediated signaling pathway / HSP90-CDC37 chaperone complex / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / aryl hydrocarbon receptor complex / histone methyltransferase binding / dynein axonemal particle / positive regulation of type 2 mitophagy / positive regulation of protein localization to cell surface / receptor ligand inhibitor activity ...regulation of type II interferon-mediated signaling pathway / HSP90-CDC37 chaperone complex / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / aryl hydrocarbon receptor complex / histone methyltransferase binding / dynein axonemal particle / positive regulation of type 2 mitophagy / positive regulation of protein localization to cell surface / receptor ligand inhibitor activity / regulation of Rho protein signal transduction / protein kinase regulator activity / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / regulation of cyclin-dependent protein serine/threonine kinase activity / ATP-dependent protein binding / Negative feedback regulation of MAPK pathway / IFNG signaling activates MAPKs / post-transcriptional regulation of gene expression / GP1b-IX-V activation signalling / telomerase holoenzyme complex assembly / Respiratory syncytial virus genome replication / Dengue virus activates/modulates innate and adaptive immune responses / ERBB2-ERBB3 signaling pathway / Drug-mediated inhibition of ERBB2 signaling / Resistance of ERBB2 KD mutants to trastuzumab / Resistance of ERBB2 KD mutants to sapitinib / Resistance of ERBB2 KD mutants to tesevatinib / Resistance of ERBB2 KD mutants to neratinib / Resistance of ERBB2 KD mutants to osimertinib / Resistance of ERBB2 KD mutants to afatinib / Resistance of ERBB2 KD mutants to AEE788 / Resistance of ERBB2 KD mutants to lapatinib / Drug resistance in ERBB2 TMD/JMD mutants / Uptake and function of diphtheria toxin / positive regulation of transforming growth factor beta receptor signaling pathway / regulation of type I interferon-mediated signaling pathway / dendritic growth cone / TPR domain binding / Assembly and release of respiratory syncytial virus (RSV) virions / neuromuscular junction development / regulation of cell differentiation / pseudopodium / Sema3A PAK dependent Axon repulsion / The NLRP3 inflammasome / regulation of protein ubiquitination / protein phosphatase activator activity / protein folding chaperone complex / HSF1-dependent transactivation / positive regulation of peptidyl-serine phosphorylation / response to unfolded protein / MAP kinase kinase kinase activity / Attenuation phase / type II interferon-mediated signaling pathway / protein targeting / HSF1 activation / chaperone-mediated protein complex assembly / Schwann cell development / axonal growth cone / RHOBTB2 GTPase cycle / negative regulation of protein-containing complex assembly / telomere maintenance via telomerase / Purinergic signaling in leishmaniasis infection / myelination / Signaling by ERBB2 / heat shock protein binding / peptide binding / insulin-like growth factor receptor signaling pathway / DNA polymerase binding / supramolecular fiber organization / protein folding chaperone / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / ESR-mediated signaling / CD209 (DC-SIGN) signaling / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Constitutive Signaling by Overexpressed ERBB2 / nitric-oxide synthase regulator activity / adenylate cyclase activator activity / ATP-dependent protein folding chaperone / wound healing / positive regulation of cell differentiation / Signaling by ERBB2 TMD/JMD mutants / Constitutive Signaling by EGFRvIII / RAF activation / Signaling by high-kinase activity BRAF mutants / Hsp90 protein binding / Signaling by ERBB2 ECD mutants / MAP2K and MAPK activation / Signaling by ERBB2 KD Mutants / Regulation of actin dynamics for phagocytic cup formation / DDX58/IFIH1-mediated induction of interferon-alpha/beta / Regulation of necroptotic cell death / tau protein binding / histone deacetylase binding / kinase binding / Stimuli-sensing channels / Downregulation of ERBB2 signaling / positive regulation of nitric oxide biosynthetic process / Chaperone Mediated Autophagy
Similarity search - Function
Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 / Cdc37, N-terminal domain ...Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 / Cdc37, N-terminal domain / Cdc37 N terminal kinase binding / 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 / Heat shock protein Hsp90, conserved site / Heat shock hsp90 proteins family signature. / C1-like domain superfamily / HSP90, C-terminal domain / Heat shock protein Hsp90, N-terminal / Heat shock protein Hsp90 family / Hsp90 protein / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / Ubiquitin-like domain superfamily / Ribosomal protein S5 domain 2-type fold / 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
ADENOSINE-5'-TRIPHOSPHATE / RAF proto-oncogene serine/threonine-protein kinase / Heat shock protein HSP 90-beta / Hsp90 co-chaperone Cdc37
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsMesa, P. / Garcia-Alonso, S. / Barbacid, M. / Montoya, G.
Funding support Denmark, 4items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
Novo Nordisk FoundationNNF0024386 Denmark
Novo Nordisk FoundationNNF17SA0030214 Denmark
Novo Nordisk FoundationNNF18OC0055061 Denmark
CitationJournal: Mol Cell / Year: 2022
Title: Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.
Authors: Sara García-Alonso / Pablo Mesa / Laura de la Puente Ovejero / Gonzalo Aizpurua / Carmen G Lechuga / Eduardo Zarzuela / Clara M Santiveri / Manuel Sanclemente / Javier Muñoz / Mónica ...Authors: Sara García-Alonso / Pablo Mesa / Laura de la Puente Ovejero / Gonzalo Aizpurua / Carmen G Lechuga / Eduardo Zarzuela / Clara M Santiveri / Manuel Sanclemente / Javier Muñoz / Mónica Musteanu / Ramón Campos-Olivas / Jorge Martínez-Torrecuadrada / Mariano Barbacid / Guillermo Montoya /
Abstract: RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full- ...RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.
History
DepositionMar 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 14, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 28, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 20, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AP1: Heat shock protein HSP 90-beta
BP1: Heat shock protein HSP 90-beta
CP1: RAF proto-oncogene serine/threonine-protein kinase
DP1: Hsp90 co-chaperone Cdc37
hetero molecules


Theoretical massNumber of molelcules
Total (without water)290,7026
Polymers289,6884
Non-polymers1,0142
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21490 Å2
ΔGint-110 kcal/mol
Surface area76090 Å2

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Components

#1: Protein Heat shock protein HSP 90-beta / HSP 90 / Heat shock 84 kDa / HSP 84 / HSP84


Mass: 84371.281 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal HA-tag + HSP90-beta / Source: (gene. exp.) Homo sapiens (human) / Gene: HSP90AB1, HSP90B, HSPC2, HSPCB / Plasmid: pcDNA3 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: P08238
#2: Protein RAF proto-oncogene serine/threonine-protein kinase / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 74409.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: RAF1 + linker Gly-Ser-Ala + C-terminal StrepTagII / Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Plasmid: pcDNA3 / Cell line (production host): Expi293F / Production host: Homo sapiens (human)
References: UniProt: P04049, non-specific serine/threonine protein kinase
#3: Protein Hsp90 co-chaperone Cdc37 / Hsp90 chaperone protein kinase-targeting subunit / p50Cdc37


Mass: 46535.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CDC37 + C-terminal Myc-DDK tag / Source: (gene. exp.) Homo sapiens (human) / Gene: CDC37, CDC37A / Plasmid: pCMV6 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q16543
#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
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: RAF1-HSP90-CDC37 complex, RHC-II / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.28913380 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F cells
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris bufferTris-HCl1
2150 mMsodium chlorideNaCl1
310 mMmagnesium chlorideMgCl21
410 mMpotassium chlorideKCl1
520 mMsodium molybdateNa2MoO41
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 3 seconds before plunging

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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 / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8138
Details: 8138 images (4430 non-tilted and 3708 30 degrees tilted)
Image scansWidth: 4096 / Height: 4096

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20rc3_4406refinement
PHENIX1.20rc3_4406refinement
EM software
IDNameVersionCategoryDetails (eV)
2EPUimage acquisition
4cryoSPARC3.3.1CTF correctionpatch CTF protocol
7UCSF ChimeraXmodel fitting
9RELION3.1.3initial Euler assignment
10RELION3.1.3final Euler assignment
12RELION3.1.33D reconstruction
13PHENIXmodel refinement
14ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3760000
Details: 3760k in total: 1353k RAF1:HSP90:CDC37 complex, 1127k 14-3-3 dimer, RAF1:14-3-3 complex 1245k
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46000 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 110.87 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008414507
ELECTRON MICROSCOPYf_angle_d0.946119501
ELECTRON MICROSCOPYf_chiral_restr0.04212136
ELECTRON MICROSCOPYf_plane_restr0.00562492
ELECTRON MICROSCOPYf_dihedral_angle_d5.3361901

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