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- EMDB-27408: Ectodomain of full-length wild-type KIT-SCF dimers -

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Entry
Database: EMDB / ID: EMD-27408
TitleEctodomain of full-length wild-type KIT-SCF dimers
Map dataEctodomain local refinement of full-length KIT-SCF dimers
Sample
  • Complex: Full-length wild-type KIT-SCF dimers reconstituted in amphipol
    • Protein or peptide: Isoform 2 of Mast/stem cell growth factor receptor Kit
    • Protein or peptide: Soluble KIT ligand
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsreceptor tyrosine kinase / cell signaling / cancer / cryo-EM / KIT / stem cell factor / oncogenic mutant / extracellular domain / asymmetric interface / structural plasticity / TRANSFERASE
Function / homology
Function and homology information


positive regulation of myeloid leukocyte differentiation / stem cell factor receptor binding / mast cell migration / positive regulation of hematopoietic progenitor cell differentiation / negative regulation of mast cell apoptotic process / positive regulation of hematopoietic stem cell proliferation / Dasatinib-resistant KIT mutants / Imatinib-resistant KIT mutants / KIT mutants bind TKIs / Masitinib-resistant KIT mutants ...positive regulation of myeloid leukocyte differentiation / stem cell factor receptor binding / mast cell migration / positive regulation of hematopoietic progenitor cell differentiation / negative regulation of mast cell apoptotic process / positive regulation of hematopoietic stem cell proliferation / Dasatinib-resistant KIT mutants / Imatinib-resistant KIT mutants / KIT mutants bind TKIs / Masitinib-resistant KIT mutants / Nilotinib-resistant KIT mutants / Regorafenib-resistant KIT mutants / Signaling by kinase domain mutants of KIT / Sunitinib-resistant KIT mutants / Signaling by juxtamembrane domain KIT mutants / Sorafenib-resistant KIT mutants / Signaling by extracellular domain mutants of KIT / stem cell factor receptor activity / hematopoietic stem cell migration / melanocyte adhesion / positive regulation of pyloric antrum smooth muscle contraction / positive regulation of colon smooth muscle contraction / positive regulation of vascular associated smooth muscle cell differentiation / melanocyte migration / Kit signaling pathway / positive regulation of dendritic cell cytokine production / regulation of bile acid metabolic process / positive regulation of small intestine smooth muscle contraction / myeloid leukocyte differentiation / positive regulation of melanocyte differentiation / mast cell differentiation / positive regulation of mast cell proliferation / mast cell chemotaxis / mast cell apoptotic process / Fc receptor signaling pathway / glycosphingolipid metabolic process / mast cell proliferation / detection of mechanical stimulus involved in sensory perception of sound / positive regulation of pseudopodium assembly / positive regulation of long-term neuronal synaptic plasticity / immature B cell differentiation / positive regulation of mast cell cytokine production / lymphoid progenitor cell differentiation / melanocyte differentiation / germ cell migration / myeloid progenitor cell differentiation / erythropoietin-mediated signaling pathway / digestive tract development / positive regulation of Ras protein signal transduction / negative regulation of programmed cell death / neural crest cell migration / positive regulation of leukocyte migration / embryonic hemopoiesis / lamellipodium assembly / tongue development / pigmentation / megakaryocyte development / Regulation of KIT signaling / stem cell population maintenance / mast cell degranulation / positive regulation of Notch signaling pathway / negative regulation of reproductive process / negative regulation of developmental process / growth factor binding / cytokine binding / somatic stem cell population maintenance / hemopoiesis / spermatid development / T cell differentiation / ectopic germ cell programmed cell death / hematopoietic progenitor cell differentiation / Transcriptional and post-translational regulation of MITF-M expression and activity / response to cadmium ion / T cell proliferation / ovarian follicle development / positive regulation of tyrosine phosphorylation of STAT protein / positive regulation of T cell proliferation / extrinsic apoptotic signaling pathway in absence of ligand / TFAP2 (AP-2) family regulates transcription of growth factors and their receptors / transmembrane receptor protein tyrosine kinase activity / SH2 domain binding / B cell differentiation / cell chemotaxis / acrosomal vesicle / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / erythrocyte differentiation / epithelial cell proliferation / filopodium / cytokine activity / stem cell differentiation / positive regulation of DNA-binding transcription factor activity / positive regulation of receptor signaling pathway via JAK-STAT / growth factor activity / visual learning / Signaling by SCF-KIT / receptor protein-tyrosine kinase / response to organic cyclic compound / fibrillar center / cytokine-mediated signaling pathway / cytoplasmic side of plasma membrane
Similarity search - Function
Stem cell factor / Stem cell factor / Mast/stem cell growth factor receptor / Tyrosine-protein kinase, receptor class III, conserved site / Receptor tyrosine kinase class III signature. / Four-helical cytokine-like, core / Immunoglobulin / Immunoglobulin domain / : / Immunoglobulin subtype 2 ...Stem cell factor / Stem cell factor / Mast/stem cell growth factor receptor / Tyrosine-protein kinase, receptor class III, conserved site / Receptor tyrosine kinase class III signature. / Four-helical cytokine-like, core / Immunoglobulin / Immunoglobulin domain / : / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Tyrosine-protein kinase, catalytic domain / Tyrosine kinase, catalytic domain / Tyrosine protein kinases specific active-site signature. / Immunoglobulin subtype / Immunoglobulin / Tyrosine-protein kinase, active site / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Mast/stem cell growth factor receptor Kit / Kit ligand
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsKrimmer SG / Bertoletti N / Mi W / Schlessinger J
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Cryo-EM analyses of KIT and oncogenic mutants reveal structural oncogenic plasticity and a target for therapeutic intervention.
Authors: Stefan G Krimmer / Nicole Bertoletti / Yoshihisa Suzuki / Luka Katic / Jyotidarsini Mohanty / Sheng Shu / Sangwon Lee / Irit Lax / Wei Mi / Joseph Schlessinger /
Abstract: The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as ...The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as acute myeloid leukemia, gastrointestinal stromal tumor, and mast cell leukemia, are driven by somatic gain-of-function KIT mutations. Here, we report cryo electron microscopy (cryo-EM) structural analyses of full-length wild-type and two oncogenic KIT mutants, which show that the overall symmetric arrangement of the extracellular domain of ligand-occupied KIT dimers contains asymmetric D5 homotypic contacts juxtaposing the plasma membrane. Mutational analysis of KIT reveals in D5 region an "Achilles heel" for therapeutic intervention. A ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stable D5 asymmetric conformation. A constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. Binding of SCF to this mutant fully restores the conformation of wild-type KIT dimers, including the formation of salt bridges responsible for D4 homotypic contacts and other hallmarks of SCF-induced KIT dimerization. These experiments reveal an unexpected structural plasticity of oncogenic KIT mutants and a therapeutic target in D5.
History
DepositionJun 22, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27408.png

Downloads & links

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Map

FileDownload / File: emd_27408.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEctodomain local refinement of full-length KIT-SCF dimers
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 450 pix.
= 605.7 Å		1.35 Å/pix.
x 450 pix.
= 605.7 Å		1.35 Å/pix.
x 450 pix.
= 605.7 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.346 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-0.78398776 - 2.6389763
Average (Standard dev.)-0.0005728361 (±0.027702885)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 605.69995 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27408_msk_1.map
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Additional map: Main map after deepEMhancer post-processing

Fileemd_27408_additional_1.map
AnnotationMain map after deepEMhancer post-processing
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Half map: Half map A of main map

Fileemd_27408_half_map_1.map
AnnotationHalf map A of main map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

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Half map: Half map B of main map

Fileemd_27408_half_map_2.map
AnnotationHalf map B of main map
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Sample components

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Entire : Full-length wild-type KIT-SCF dimers reconstituted in amphipol

EntireName: Full-length wild-type KIT-SCF dimers reconstituted in amphipol
Components
  • Complex: Full-length wild-type KIT-SCF dimers reconstituted in amphipol
    • Protein or peptide: Isoform 2 of Mast/stem cell growth factor receptor Kit
    • Protein or peptide: Soluble KIT ligand
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Full-length wild-type KIT-SCF dimers reconstituted in amphipol

SupramoleculeName: Full-length wild-type KIT-SCF dimers reconstituted in amphipol
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 250 KDa

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Macromolecule #1: Isoform 2 of Mast/stem cell growth factor receptor Kit

MacromoleculeName: Isoform 2 of Mast/stem cell growth factor receptor Kit
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: receptor protein-tyrosine kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 110.657008 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MTILCWLALL STLTAVNADY KDDDDKRPHA MGEPSPPSIH PGKSDLIVRV GDEIRLLCTD PGFVKWTFEI LDETNENKQN EWITEKAEA TNTGKYTCTN KHGLSNSIYV FVRDPAKLFL VDRSLYGKED NDTLVRCPLT DPEVTNYSLK GCQGKPLPKD L RFIPDPKA ...String:
MTILCWLALL STLTAVNADY KDDDDKRPHA MGEPSPPSIH PGKSDLIVRV GDEIRLLCTD PGFVKWTFEI LDETNENKQN EWITEKAEA TNTGKYTCTN KHGLSNSIYV FVRDPAKLFL VDRSLYGKED NDTLVRCPLT DPEVTNYSLK GCQGKPLPKD L RFIPDPKA GIMIKSVKRA YHRLCLHCSV DQEGKSVLSE KFILKVRPAF KAVPVVSVSK ASYLLREGEE FTVTCTIKDV SS SVYSTWK RENSQTKLQE KYNSWHHGDF NYERQATLTI SSARVNDSGV FMCYANNTFG SANVTTTLEV VDKGFINIFP MIN TTVFVN DGENVDLIVE YEAFPKPEHQ QWIYMNRTFT DKWEDYPKSE NESNIRYVSE LHLTRLKGTE GGTYTFLVSN SDVN AAIAF NVYVNTKPEI LTYDRLVNGM LQCVAAGFPE PTIDWYFCPG TEQRCSASVL PVDVQTLNSS GPPFGKLVVQ SSIDS SAFK HNGTVECKAY NDVGKTSAYF NFAFKEQIHP HTLFTPLLIG FVIVAGMMCI IVMILTYKYL QKPMYEVQWK VVEEIN GNN YVYIDPTQLP YDHKWEFPRN RLSFGKTLGA GAFGKVVEAT AYGLIKSDAA MTVAVAMLKP SAHLTEREAL MSELKVL SY LGNHMNIVNL LGACTIGGPT LVITEYCCYG DLLNFLRRKR DSFICSKQED HAEAALYKNL LHSKESSCSD STNEYMDM K PGVSYVVPTK ADKRRSVRIG SYIERDVTPA IMEDDELALD LEDLLSFSYQ VAKGMAFLAS KNCIHRDLAA RNILLTHGR ITKICDFGLA RDIKNDSNYV VKGNARLPVK WMAPESIFNC VYTFESDVWS YGIFLWELFS LGSSPYPGMP VDSKFYKMIK EGFRMLSPE HAPAEMYDIM KTCWDADPLK RPTFKQIVQL IEKQISESTN HIYSNLANCS PNRQKPVVDH SVRINSVGST A SSSQPLLV HDDVGSHHHH HH

UniProtKB: Mast/stem cell growth factor receptor Kit

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Macromolecule #2: Soluble KIT ligand

MacromoleculeName: Soluble KIT ligand / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 16.007306 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
EGICRNRVTN NVKDVTKLVA NLPKDYMITL KYVPGMDVLP SHCWISEMVV QLSDSLTDLL DKFSNISEGL SNYSIIDKLV NIVDDLVEC VKENSSKDLK KSFKSPEPRL FTPEEFFRIF NRSIDAFKDF VVASETSDCV VS

UniProtKB: Kit ligand

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration5.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4S(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
200.0 mMNaClSodium chloride
0.1 %C13H17F13NO4P(1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: GATAN CRYOPLUNGE 3 / Details: Blotting time 3 sec, blotting force 0..
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsSerialEM COMA-FREE ALIGNMENT
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5925 / Average exposure time: 8.9 sec. / Average electron dose: 49.23 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1096589
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1.0) / Number images used: 500569
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.1.0)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 3.1.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2e9w


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 117
Output model

PDB-8dfm:
Ectodomain of full-length wild-type KIT-SCF dimers

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