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- EMDB-43931: CryoEM structure of activated CRAF/MEK/14-3-3 complex with NST-628 -

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Entry
Database: EMDB / ID: EMD-43931
TitleCryoEM structure of activated CRAF/MEK/14-3-3 complex with NST-628
Map data
Sample
  • Complex: CRAF/MEK/14-3-3 complex with NST-628
    • Protein or peptide: GST26/CRAF chimera
    • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
    • Protein or peptide: 14-3-3 protein sigma
  • Ligand: 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
KeywordsInhibitor / complex / SIGNALING PROTEIN / TRANSFERASE
Function / homology
Function and homology information


death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / negative regulation of homotypic cell-cell adhesion / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / regulation of vascular associated smooth muscle contraction / intermediate filament cytoskeleton organization / mitogen-activated protein kinase kinase / Golgi inheritance / placenta blood vessel development ...death-inducing signaling complex assembly / epithelial cell proliferation involved in lung morphogenesis / positive regulation of endodermal cell differentiation / negative regulation of homotypic cell-cell adhesion / negative regulation of hypoxia-induced intrinsic apoptotic signaling pathway / regulation of vascular associated smooth muscle contraction / intermediate filament cytoskeleton organization / mitogen-activated protein kinase kinase / Golgi inheritance / placenta blood vessel development / MAP-kinase scaffold activity / positive regulation of muscle contraction / regulation of axon regeneration / cerebellar cortex formation / labyrinthine layer development / regulation of Rho protein signal transduction / melanosome transport / type B pancreatic cell proliferation / Signaling by MAP2K mutants / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / vesicle transport along microtubule / positive regulation of axonogenesis / positive regulation of Ras protein signal transduction / insulin secretion involved in cellular response to glucose stimulus / regulation of Golgi inheritance / mitogen-activated protein kinase kinase kinase binding / central nervous system neuron differentiation / triglyceride homeostasis / trachea formation / Negative feedback regulation of MAPK pathway / regulation of early endosome to late endosome transport / IFNG signaling activates MAPKs / regulation of stress-activated MAPK cascade / Frs2-mediated activation / GP1b-IX-V activation signalling / regulation of epidermal cell division / MAPK3 (ERK1) activation / protein kinase C inhibitor activity / ERBB2-ERBB3 signaling pathway / positive regulation of epidermal cell differentiation / keratinocyte development / keratinization / neurotrophin TRK receptor signaling pathway / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / pseudopodium / face development / endodermal cell differentiation / regulation of cell-cell adhesion / MAP kinase kinase activity / Bergmann glial cell differentiation / positive regulation of ATP biosynthetic process / regulation of cell differentiation / thyroid gland development / glutathione transferase / Uptake and function of anthrax toxins / cAMP/PKA signal transduction / Regulation of localization of FOXO transcription factors / keratinocyte proliferation / glutathione transferase activity / positive regulation of protein serine/threonine kinase activity / extrinsic apoptotic signaling pathway via death domain receptors / somatic stem cell population maintenance / protein kinase activator activity / positive regulation of peptidyl-serine phosphorylation / phosphoserine residue binding / Activation of BAD and translocation to mitochondria / MAP kinase kinase kinase activity / negative regulation of keratinocyte proliferation / establishment of skin barrier / type II interferon-mediated signaling pathway / negative regulation of protein localization to plasma membrane / negative regulation of protein-containing complex assembly / Schwann cell development / response to axon injury / Chk1/Chk2(Cds1) mediated inactivation of Cyclin B:Cdk1 complex / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / negative regulation of protein kinase activity / negative regulation of stem cell proliferation / keratinocyte differentiation / SARS-CoV-1 targets host intracellular signalling and regulatory pathways / RHO GTPases activate PKNs / neuron projection morphogenesis / positive regulation of protein localization / response to muscle stretch / ERK1 and ERK2 cascade / myelination / glutathione metabolic process / positive regulation of autophagy / protein serine/threonine/tyrosine kinase activity / CD209 (DC-SIGN) signaling / positive regulation of cell adhesion / dendrite cytoplasm / insulin-like growth factor receptor signaling pathway / protein sequestering activity / protein export from nucleus / negative regulation of innate immune response / response to glucocorticoid
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 / Glutathione S-transferase, N-terminal domain / 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 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.36 Å
AuthorsQuade B / Cohen SE / Huang X
Funding support1 items
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, 2024-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43931.png

Downloads & links

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Map

FileDownload / File: emd_43931.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.81 Å/pix.
x 200 pix.
= 362.1 Å		1.81 Å/pix.
x 200 pix.
= 362.1 Å		1.81 Å/pix.
x 200 pix.
= 362.1 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.8105 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0464
Minimum - Maximum-0.10732846 - 0.20123623
Average (Standard dev.)0.00022246753 (±0.0045600175)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 362.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43931_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_43931_half_map_2.map
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Histogram (log scale)

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Sample components

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Entire : CRAF/MEK/14-3-3 complex with NST-628

EntireName: CRAF/MEK/14-3-3 complex with NST-628
Components
  • Complex: CRAF/MEK/14-3-3 complex with NST-628
    • Protein or peptide: GST26/CRAF chimera
    • Protein or peptide: Dual specificity mitogen-activated protein kinase kinase 1
    • Protein or peptide: 14-3-3 protein sigma
  • Ligand: 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

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Supramolecule #1: CRAF/MEK/14-3-3 complex with NST-628

SupramoleculeName: CRAF/MEK/14-3-3 complex with NST-628 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: GST26/CRAF chimera

MacromoleculeName: GST26/CRAF chimera / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: glutathione transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 64.798465 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK WRNKKFELGL EFPNLPYYID GDVKLTQSMA IIRYIADKHN MLGGCPKER AEISMLEGAV LDIRYGVSRI AYSKDFETLK VDFLSKLPEM LKMFEDRLCH KTYLNGDHVT HPDFMLYDAL D VVLYMDPM ...String:
MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK WRNKKFELGL EFPNLPYYID GDVKLTQSMA IIRYIADKHN MLGGCPKER AEISMLEGAV LDIRYGVSRI AYSKDFETLK VDFLSKLPEM LKMFEDRLCH KTYLNGDHVT HPDFMLYDAL D VVLYMDPM CLDAFPKLVC FKKRIEAIPQ IDKYLKSSKY IAWPLQGWQA TFGGGDHPPK SDSQPKTPVP AQRERAPVSG TQ EKNKIRP RGQRDSSDDW EIEASEVMLS TRIGSGSFGT VYKGKWHGDV AVKILKVVDP TPEQFQAFRN EVAVLRKTRH VNI LLFMGY MTKDNLAIVT QWCEGSSLYK HLHVQETKFQ MFQLIDIARQ TAQGMDYLHA KNIIHRDMKS NNIFLHEGLT VKIG DFGLA TVKSRWSGSQ QVEQPTGSVL WMAPEVIRMQ DNNPFSFQSD VYSYGIVLYE LMTGELPYSH INNRDQIIFM VGRGY ASPD LSKLYKNCPK AMKRLVADCV KKVKEERPLF PQILSSIELL QHSLPKINRS A(SEP)EPSLHRAA HTEDINACTL TT SPRLPVF

UniProtKB: Glutathione S-transferase class-mu 26 kDa isozyme, RAF proto-oncogene serine/threonine-protein kinase

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Macromolecule #2: Dual specificity mitogen-activated protein kinase kinase 1

MacromoleculeName: Dual specificity mitogen-activated protein kinase kinase 1
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: mitogen-activated protein kinase kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.518988 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GMPKKKPTPI QLNPAPDGSA VNGTSSAETN LEALQKKLEE LELDEQQRKR LEAFLTQKQK VGELKDDDFE KISELGAGNG GVVFKVSHK PSGLVMARKL IHLEIKPAIR NQIIRELQVL HECNSPYIVG FYGAFYSDGE ISICMEHMDG GSLDQVLKKA G RIPEQILG ...String:
GMPKKKPTPI QLNPAPDGSA VNGTSSAETN LEALQKKLEE LELDEQQRKR LEAFLTQKQK VGELKDDDFE KISELGAGNG GVVFKVSHK PSGLVMARKL IHLEIKPAIR NQIIRELQVL HECNSPYIVG FYGAFYSDGE ISICMEHMDG GSLDQVLKKA G RIPEQILG KVSIAVIKGL TYLREKHKIM HRDVKPSNIL VNSRGEIKLC DFGVSGQLID AMANAFVGTR SYMSPERLQG TH YSVQSDI WSMGLSLVEM AVGRYPIPPP DAKELELMFG CQVEGDAAET PPRPRTPGRP LSSYGMDSRP PMAIFELLDY IVN EPPPKL PSGVFSLEFQ DFVNKCLIKN PAERADLKQL MVHAFIKRSD AEEVDFAGWL CSTIGLNQPS TPTHAAGV

UniProtKB: Dual specificity mitogen-activated protein kinase kinase 1

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Macromolecule #3: 14-3-3 protein sigma

MacromoleculeName: 14-3-3 protein sigma / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.807064 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MERASLIQKA KLAEQAERYE DMAAFMKGAV EKGEELSCEE RNLLSVAYKN VVGGQRAAWR VLSSIEQKSN EEGSEEKGPE VREYREKVE TELQGVCDTV LGLLDSHLIK EAGDAESRVF YLKMKGDYYR YLAEVATGDD KKRIIDSARS AYQEAMDISK K EMPPTNPI ...String:
MERASLIQKA KLAEQAERYE DMAAFMKGAV EKGEELSCEE RNLLSVAYKN VVGGQRAAWR VLSSIEQKSN EEGSEEKGPE VREYREKVE TELQGVCDTV LGLLDSHLIK EAGDAESRVF YLKMKGDYYR YLAEVATGDD KKRIIDSARS AYQEAMDISK K EMPPTNPI RLGLALNFSV FHYEIANSPE EAISLAKTTF DEAMADLHTL SEDSYKDSTL IMQLLRDNLT LWTADNAGEE GG EAPQEPQ S

UniProtKB: 14-3-3 protein sigma

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Macromolecule #4: N-[3-fluoro-4-({7-[(3-fluoropyridin-2-yl)oxy]-4-methyl-2-oxo-2H-1...

MacromoleculeName: 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
type: ligand / ID: 4 / Number of copies: 2 / Formula: A1AHE
Molecular weightTheoretical: 488.464 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

20550

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.36 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 83248
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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