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- PDB-6xar: Structure of CBL tyrosine kinase binding domain (TKBD) with C-ter... -

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Basic information

Entry
Database: PDB / ID: 6xar
TitleStructure of CBL tyrosine kinase binding domain (TKBD) with C-terminal tail of Src-like kinase protein 2 (SLAP2)
Components
  • E3 ubiquitin-protein ligase CBL
  • Src-like-adapter 2
KeywordsSIGNALING PROTEIN / adaptor / E3 ubiquitin ligase / activation / complex
Function / homology
Function and homology information


Negative regulation of FLT3 / negative regulation of calcium-mediated signaling / regulation of platelet-derived growth factor receptor-alpha signaling pathway / ubiquitin-dependent endocytosis / regulation of Rap protein signal transduction / : / entry of bacterium into host cell / flotillin complex / 1-phosphatidylinositol-3-kinase regulator activity / phosphatidylinositol 3-kinase regulatory subunit binding ...Negative regulation of FLT3 / negative regulation of calcium-mediated signaling / regulation of platelet-derived growth factor receptor-alpha signaling pathway / ubiquitin-dependent endocytosis / regulation of Rap protein signal transduction / : / entry of bacterium into host cell / flotillin complex / 1-phosphatidylinositol-3-kinase regulator activity / phosphatidylinositol 3-kinase regulatory subunit binding / phosphatidylinositol 3-kinase complex / positive regulation of epidermal growth factor receptor signaling pathway / negative regulation of T cell receptor signaling pathway / Regulation of KIT signaling / mast cell degranulation / Interleukin-6 signaling / response to testosterone / cellular response to platelet-derived growth factor stimulus / negative regulation of epidermal growth factor receptor signaling pathway / TGF-beta receptor signaling activates SMADs / response to starvation / protein monoubiquitination / phosphatidylinositol phosphate biosynthetic process / protein autoubiquitination / signaling adaptor activity / FLT3 signaling by CBL mutants / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Negative regulation of FLT3 / T cell activation / ephrin receptor binding / phosphotyrosine residue binding / InlB-mediated entry of Listeria monocytogenes into host cell / cellular response to nerve growth factor stimulus / response to activity / Regulation of signaling by CBL / response to gamma radiation / Negative regulation of FGFR3 signaling / Negative regulation of FGFR2 signaling / EGFR downregulation / Negative regulation of FGFR4 signaling / Negative regulation of FGFR1 signaling / Spry regulation of FGF signaling / Constitutive Signaling by EGFRvIII / RING-type E3 ubiquitin transferase / Negative regulation of MET activity / receptor tyrosine kinase binding / cytokine-mediated signaling pathway / cilium / positive regulation of receptor-mediated endocytosis / SH3 domain binding / protein polyubiquitination / ubiquitin-protein transferase activity / Signaling by CSF1 (M-CSF) in myeloid cells / male gonad development / ubiquitin protein ligase activity / late endosome / Cargo recognition for clathrin-mediated endocytosis / Constitutive Signaling by Ligand-Responsive EGFR Cancer Variants / Clathrin-mediated endocytosis / growth cone / cellular response to hypoxia / cytoplasmic vesicle / ubiquitin-dependent protein catabolic process / response to ethanol / adaptive immune response / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / protein ubiquitination / cadherin binding / membrane raft / intracellular membrane-bounded organelle / focal adhesion / DNA damage response / calcium ion binding / negative regulation of apoptotic process / perinuclear region of cytoplasm / Golgi apparatus / negative regulation of transcription by RNA polymerase II / signal transduction / nucleoplasm / plasma membrane / cytosol
Similarity search - Function
Src-like-adapter 2 / SLAP, SH2 domain / Adaptor protein Cbl, N-terminal helical / Adaptor protein Cbl, EF hand-like / Adaptor protein Cbl, SH2-like domain / Adaptor protein Cbl, PTB domain / Adaptor protein Cbl / CBL proto-oncogene N-terminal domain 1 / CBL proto-oncogene N-terminus, EF hand-like domain / CBL proto-oncogene N-terminus, SH2-like domain ...Src-like-adapter 2 / SLAP, SH2 domain / Adaptor protein Cbl, N-terminal helical / Adaptor protein Cbl, EF hand-like / Adaptor protein Cbl, SH2-like domain / Adaptor protein Cbl, PTB domain / Adaptor protein Cbl / CBL proto-oncogene N-terminal domain 1 / CBL proto-oncogene N-terminus, EF hand-like domain / CBL proto-oncogene N-terminus, SH2-like domain / Cbl-type phosphotyrosine-binding (Cbl-PTB) domain profile. / Adaptor protein Cbl, N-terminal domain superfamily / UBA/TS-N domain / Ubiquitin associated domain / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / Ubiquitin-associated domain / Ubiquitin-associated domain (UBA) profile. / UBA-like superfamily / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / EF-hand domain pair / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
E3 ubiquitin-protein ligase CBL / Src-like-adapter 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.501 Å
AuthorsWybenga-Groot, L.E. / McGlade, C.J.
Funding support Canada, 2items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)MOP-12859 Canada
Canadian Institutes of Health Research (CIHR)FRN166034 Canada
Citation
Journal: J.Mol.Biol. / Year: 2021
Title: SLAP2 Adaptor Binding Disrupts c-CBL Autoinhibition to Activate Ubiquitin Ligase Function.
Authors: Wybenga-Groot, L.E. / Tench, A.J. / Simpson, C.D. / Germain, J.S. / Raught, B. / Moran, M.F. / McGlade, C.J.
#1: Journal: Acta Crystallogr.,Sect.F / Year: 2021
Title: Sleuthing biochemical evidence to elucidate unassigned electron density in a CBL-SLAP2 crystal complex
Authors: Wybenga-Groot, L.E. / McGlade, C.J.
History
DepositionJun 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 3, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: E3 ubiquitin-protein ligase CBL
B: E3 ubiquitin-protein ligase CBL
C: Src-like-adapter 2
D: Src-like-adapter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)81,8806
Polymers81,8004
Non-polymers802
Water84747
1
A: E3 ubiquitin-protein ligase CBL
C: Src-like-adapter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,9403
Polymers40,9002
Non-polymers401
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1430 Å2
ΔGint-25 kcal/mol
Surface area15430 Å2
MethodPISA
2
B: E3 ubiquitin-protein ligase CBL
D: Src-like-adapter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,9403
Polymers40,9002
Non-polymers401
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1480 Å2
ΔGint-25 kcal/mol
Surface area14760 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.730, 86.960, 65.260
Angle α, β, γ (deg.)90.000, 112.310, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein E3 ubiquitin-protein ligase CBL / Casitas B-lineage lymphoma proto-oncogene / Proto-oncogene c-Cbl / RING finger protein 55 / RING- ...Casitas B-lineage lymphoma proto-oncogene / Proto-oncogene c-Cbl / RING finger protein 55 / RING-type E3 ubiquitin transferase CBL / Signal transduction protein CBL


Mass: 38832.828 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CBL, CBL2, RNF55 / Plasmid: GST-CBL / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: P22681, RING-type E3 ubiquitin transferase
#2: Protein/peptide Src-like-adapter 2 / Src-like adapter protein 2 / SLAP-2


Mass: 2067.234 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Sla2 / Plasmid: Trx-His(6)-mSLAP2 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q8R4L0-2
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 47 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Sequence detailsThe mouse SLAP2 construct was degraded during crystallization. The originally expressed sequence, ...The mouse SLAP2 construct was degraded during crystallization. The originally expressed sequence, after tag cleavage, was GSQPERHKVTAVALGSFPAGEQARLSLRLGEPLTIISEDGDWWTVQSEVSGREYHMPSVY VAKVAHGWLYEGLSREKAEELLLLPGNPGGAFLIRESQTRRGCYSLSVRLSRPASWDRIR HYRIQRLDNGWLYISPRLTFPSLHALVEHYSELADGICCPLREPCVLQKLGPLPGKDTPP PVTVPTSSLNWKKLDRSLLFLEAPASGEASLLSEGLRESLSSYISLAEDPLDDA corresponding to residues 28-259 of SLAP2. Only residues 237-255 were observed in the electron density with the remainder presumably degraded.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.01 Å3/Da / Density % sol: 38.9 % / Description: rod-like
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: CBL/SLAP2 complex mixed in hanging drop with equal volume (200 nL) of 0.1 M Hepes pH 7.5, 10% (w/v) PEG8000
Temp details: room temperature

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.97921 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Mar 22, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97921 Å / Relative weight: 1
ReflectionResolution: 2.5→45.323 Å / Num. obs: 20374 / % possible obs: 90.5 % / Redundancy: 2.9 % / Biso Wilson estimate: 40.17 Å2 / CC1/2: 0.993 / Rmerge(I) obs: 0.08 / Rpim(I) all: 0.055 / Rrim(I) all: 0.098 / Net I/σ(I): 8.3
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.5-2.620.601283314470.570.530.8061.557.8
9.01-45.322.80.03913404860.9950.0270.04718.396.2

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation2.5 Å45.32 Å
Translation2.5 Å45.32 Å

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Processing

Software
NameVersionClassification
MOSFLMdata reduction
Aimless0.3.11data scaling
PHASER2.5.7phasing
PHENIXdevrefinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1B47

1b47


Resolution: 2.501→45.323 Å / SU ML: 0.42 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 33.2 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2934 943 5 %
Rwork0.2493 17926 -
obs0.2515 18869 83.81 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 112.68 Å2 / Biso mean: 48.5193 Å2 / Biso min: 16.3 Å2
Refinement stepCycle: final / Resolution: 2.501→45.323 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4954 0 2 47 5003
Biso mean--49.6 35.81 -
Num. residues----643
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0025085
X-RAY DIFFRACTIONf_angle_d0.6026918
X-RAY DIFFRACTIONf_chiral_restr0.027774
X-RAY DIFFRACTIONf_plane_restr0.003880
X-RAY DIFFRACTIONf_dihedral_angle_d10.9731778
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.501-2.63240.3507490.310477126
2.6324-2.79730.3342960.3224200366
2.7973-3.01330.45071640.3374290696
3.0133-3.31640.34221570.29053060100
3.3164-3.79610.28221300.25363079100
3.7961-4.78190.25781810.21153042100
4.7819-45.3230.2451660.213306598

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