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- PDB-9axa: CryoEM structure of activated CRAF/MEK/14-3-3 complex with NST-628 -

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Basic information

Entry
Database: PDB / ID: 9axa
TitleCryoEM structure of activated CRAF/MEK/14-3-3 complex with NST-628
Components
  • 14-3-3 protein sigma
  • Dual specificity mitogen-activated protein kinase kinase 1
  • GST26/CRAF chimera
KeywordsTRANSFERASE / Inhibitor / complex / SIGNALING PROTEIN
Function / homology
Function and homology information


death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / JUN kinase kinase activity / regulation of axon regeneration / mitogen-activated protein kinase kinase / placenta blood vessel development / MAP-kinase scaffold activity / labyrinthine layer development ...death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / intermediate filament cytoskeleton organization / JUN kinase kinase activity / regulation of axon regeneration / mitogen-activated protein kinase kinase / placenta blood vessel development / MAP-kinase scaffold activity / labyrinthine layer development / cerebellar cortex formation / type B pancreatic cell proliferation / regulation of Rho protein signal transduction / Signaling by MAP2K mutants / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / positive regulation of axonogenesis / insulin secretion involved in cellular response to glucose stimulus / regulation of Golgi inheritance / trachea formation / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / regulation of stress-activated MAPK cascade / IFNG signaling activates MAPKs / MAPK3 (ERK1) activation / GP1b-IX-V activation signalling / Frs2-mediated activation / regulation of epidermal cell division / protein kinase C inhibitor activity / positive regulation of epidermal cell differentiation / keratinocyte development / ERBB2-ERBB3 signaling pathway / keratinization / endodermal cell differentiation / regulation of cell-cell adhesion / face development / MAP kinase kinase activity / pseudopodium / neurotrophin TRK receptor signaling pathway / Bergmann glial cell differentiation / regulation of cell differentiation / Uptake and function of anthrax toxins / glutathione transferase / thyroid gland development / cAMP/PKA signal transduction / Regulation of localization of FOXO transcription factors / somatic stem cell population maintenance / protein kinase activator activity / keratinocyte proliferation / extrinsic apoptotic signaling pathway via death domain receptors / glutathione transferase activity / phosphoserine residue binding / MAP kinase kinase kinase activity / Activation of BAD and translocation to mitochondria / type II interferon-mediated signaling pathway / negative regulation of keratinocyte proliferation / positive regulation of protein serine/threonine kinase activity / establishment of skin barrier / negative regulation of protein localization to plasma membrane / negative regulation of protein-containing complex assembly / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / Schwann cell development / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / negative regulation of stem cell proliferation / keratinocyte differentiation / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / RHO GTPases activate PKNs / activation of adenylate cyclase activity / positive regulation of peptidyl-serine phosphorylation / positive regulation of protein localization / response to muscle stretch / myelination / ERK1 and ERK2 cascade / CD209 (DC-SIGN) signaling / glutathione metabolic process / protein serine/threonine/tyrosine kinase activity / positive regulation of cell adhesion / protein sequestering activity / insulin-like growth factor receptor signaling pathway / negative regulation of innate immune response / protein export from nucleus / release of cytochrome c from mitochondria / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / protein serine/threonine kinase activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / thymus development / positive regulation of protein export from nucleus / negative regulation of protein kinase activity / Signal transduction by L1 / stem cell proliferation / Translocation of SLC2A4 (GLUT4) to the plasma membrane / TP53 Regulates Metabolic Genes / cell motility / RAF activation / wound healing / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / Stimuli-sensing channels
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 / Glutathione S-transferase, C-terminal domain / : / : / 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 / Glutathione S-transferase, C-terminal domain / : / : / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / 14-3-3 protein sigma / 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 / Glutathione S-transferase, N-terminal domain / Glutathione transferase family / Glutathione S-transferase, C-terminal / 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 / Glutathione S-transferase, C-terminal-like / Soluble glutathione S-transferase C-terminal domain profile. / Soluble glutathione S-transferase N-terminal domain profile. / Glutathione S-transferase, N-terminal / Glutathione S-transferase, C-terminal domain superfamily / Thioredoxin-like superfamily / 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
Similarity search - Domain/homology
: / RAF proto-oncogene serine/threonine-protein kinase / Glutathione S-transferase class-mu 26 kDa isozyme / 14-3-3 protein sigma / Dual specificity mitogen-activated protein kinase kinase 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.36 Å
AuthorsQuade, B. / Cohen, S.E. / Huang, X.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cancer Discov / Year: 2024
Title: The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers.
Authors: Meagan B Ryan / Bradley Quade / Natasha Schenk / Zhong Fang / Marshall Zingg / Steven E Cohen / Brooke M Swalm / Chun Li / Ayşegül Özen / Chaoyang Ye / Maria Stella Ritorto / Xin Huang / ...Authors: Meagan B Ryan / Bradley Quade / Natasha Schenk / Zhong Fang / Marshall Zingg / Steven E Cohen / Brooke M Swalm / Chun Li / Ayşegül Özen / Chaoyang Ye / Maria Stella Ritorto / Xin Huang / Arvin C Dar / Yongxin Han / Klaus P Hoeflich / Michael Hale / Margit Hagel /
Abstract: Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the ...Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.
History
DepositionMar 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _em_admin.last_update
Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: 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
A: GST26/CRAF chimera
B: Dual specificity mitogen-activated protein kinase kinase 1
C: GST26/CRAF chimera
D: Dual specificity mitogen-activated protein kinase kinase 1
E: 14-3-3 protein sigma
F: 14-3-3 protein sigma
hetero molecules


Theoretical massNumber of molelcules
Total (without water)273,2268
Polymers272,2496
Non-polymers9772
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein GST26/CRAF chimera / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 64798.465 Da / Num. of mol.: 2 / Mutation: Y340D Y341D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Production host: Homo sapiens (human)
References: UniProt: P08515, UniProt: P04049, glutathione transferase, 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: 43518.988 Da / Num. of mol.: 2 / Mutation: S218A S222A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAP2K1, MEK1, PRKMK1 / Production host: Homo sapiens (human)
References: UniProt: Q02750, mitogen-activated protein kinase kinase
#3: Protein 14-3-3 protein sigma / Epithelial cell marker protein 1 / Stratifin


Mass: 27807.064 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SFN, HME1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P31947
#4: Chemical ChemComp-A1AHE / N-[3-fluoro-4-({7-[(3-fluoropyridin-2-yl)oxy]-4-methyl-2-oxo-2H-1-benzopyran-3-yl}methyl)pyridin-2-yl]-N'-methylsulfuric diamide


Mass: 488.464 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H18F2N4O5S / 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: CRAF/MEK/14-3-3 complex with NST-628 / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83248 / Symmetry type: POINT
RefinementCross valid method: NONE

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