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- PDB-8dfp: Ectodomain of full-length KIT(DupA502,Y503)-SCF dimers -

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Basic information

Entry
Database: PDB / ID: 8dfp
TitleEctodomain of full-length KIT(DupA502,Y503)-SCF dimers
Components
  • Isoform 2 of Mast/stem cell growth factor receptor Kit
  • Soluble KIT ligand
KeywordsTRANSFERASE / receptor tyrosine kinase / cell signaling / cancer / cryo-EM / KIT / stem cell factor / oncogenic mutant / extracellular domain / asymmetric interface / structural plasticity
Function / homology
Function and homology information


positive regulation of myeloid leukocyte differentiation / negative regulation of mast cell apoptotic process / stem cell factor receptor binding / mast cell migration / positive regulation of hematopoietic progenitor cell differentiation / 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 / negative regulation of mast cell apoptotic process / stem cell factor receptor binding / mast cell migration / positive regulation of hematopoietic progenitor cell differentiation / 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 / erythropoietin-mediated signaling pathway / positive regulation of vascular associated smooth muscle cell differentiation / melanocyte migration / positive regulation of dendritic cell cytokine production / Kit signaling pathway / positive regulation of melanocyte differentiation / regulation of bile acid metabolic process / positive regulation of small intestine smooth muscle contraction / mast cell apoptotic process / mast cell differentiation / positive regulation of mast cell proliferation / mast cell chemotaxis / myeloid leukocyte differentiation / Fc receptor signaling pathway / glycosphingolipid metabolic process / mast cell proliferation / positive regulation of long-term neuronal synaptic plasticity / detection of mechanical stimulus involved in sensory perception of sound / positive regulation of pseudopodium assembly / positive regulation of mast cell cytokine production / immature B cell differentiation / melanocyte differentiation / germ cell migration / lymphoid progenitor cell differentiation / myeloid progenitor cell differentiation / digestive tract development / positive regulation of Ras protein signal transduction / negative regulation of programmed cell death / neural crest cell migration / embryonic hemopoiesis / lamellipodium assembly / pigmentation / positive regulation of leukocyte migration / tongue development / megakaryocyte development / Regulation of KIT signaling / mast cell degranulation / stem cell population maintenance / positive regulation of Notch signaling pathway / cytokine binding / negative regulation of reproductive process / negative regulation of developmental process / spermatid development / growth factor binding / somatic stem cell population maintenance / hemopoiesis / T cell differentiation / ectopic germ cell programmed cell death / hematopoietic progenitor cell differentiation / positive regulation of phospholipase C activity / response to cadmium ion / T cell proliferation / ovarian follicle development / positive regulation of tyrosine phosphorylation of STAT protein / extrinsic apoptotic signaling pathway in absence of ligand / positive regulation of T cell proliferation / TFAP2 (AP-2) family regulates transcription of growth factors and their receptors / transmembrane receptor protein tyrosine kinase activity / SH2 domain binding / cell chemotaxis / B cell differentiation / erythrocyte differentiation / acrosomal vesicle / Signaling by phosphorylated juxtamembrane, extracellular and kinase domain KIT mutants / filopodium / epithelial cell proliferation / cytokine activity / stem cell differentiation / positive regulation of receptor signaling pathway via JAK-STAT / growth factor activity / visual learning / Signaling by SCF-KIT / cytoplasmic side of plasma membrane / response to organic cyclic compound / receptor protein-tyrosine kinase / fibrillar center / cytokine-mediated signaling pathway / male gonad development
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 / Immunoglobulin C-2 Type ...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 / Protein tyrosine and serine/threonine kinase / Serine-threonine/tyrosine-protein kinase, catalytic domain / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsBertoletti, N. / Krimmer, S.G. / Mi, W. / Schlessinger, J.
Funding support1items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform 2 of Mast/stem cell growth factor receptor Kit
B: Isoform 2 of Mast/stem cell growth factor receptor Kit
C: Soluble KIT ligand
D: Soluble KIT ligand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)253,40810
Polymers251,2684
Non-polymers2,1406
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Isoform 2 of Mast/stem cell growth factor receptor Kit / SCFR / Piebald trait protein / PBT / Proto-oncogene c-Kit / Tyrosine-protein kinase Kit / p145 c- ...SCFR / Piebald trait protein / PBT / Proto-oncogene c-Kit / Tyrosine-protein kinase Kit / p145 c-kit / v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog


Mass: 110891.258 Da / Num. of mol.: 2 / Mutation: K619A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KIT, SCFR / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P10721, receptor protein-tyrosine kinase
#2: Protein Soluble KIT ligand / sKITLG


Mass: 14742.981 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KITLG, MGF, SCF / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): CodonPlus RIPL / References: UniProt: P21583
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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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: Full-length KIT(DupA502,Y503)-SCF dimers reconstituted in amphipol
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human) / Cellular location: membrane
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: Bacmid
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)C8H18N2O4S1
2200 mMSodium chlorideNaClSodium chloride1
30.1 %(1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholineC13H17F13NO4P1
SpecimenConc.: 5.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 K / Details: Blotting time 3 sec, blotting force 0.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: SerialEM COMA-FREE ALIGNMENT
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 11.134 sec. / Electron dose: 64.52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8456

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.2.0particle selection
2SerialEMimage acquisition
4cryoSPARC3.2.0CTF correction
7ISOLDE1.0b3model fitting
9cryoSPARC3.2.0initial Euler assignment
10cryoSPARC3.2.0final Euler assignment
11cryoSPARC3.2.0classification
12cryoSPARC3.2.03D reconstruction
13PHENIX1.02.1-4487-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3649954
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 865760 / Symmetry type: POINT
Atomic model buildingB value: 119.4 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 8DFM

8dfm

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316317
ELECTRON MICROSCOPYf_angle_d0.88129285
ELECTRON MICROSCOPYf_dihedral_angle_d15.4844462
ELECTRON MICROSCOPYf_chiral_restr0.071439
ELECTRON MICROSCOPYf_plane_restr0.0092534

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