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- EMDB-47134: CHIP U-box dimer bound to Fab 2F1 -

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Basic information

Entry
Database: EMDB / ID: EMD-47134
TitleCHIP U-box dimer bound to Fab 2F1
Map dataSharpened Map
Sample
  • Complex: CHIP U-box dimer bound to Fab 2F1
    • Protein or peptide: E3 ubiquitin-protein ligase CHIP
    • Protein or peptide: 2F1 Fab heavy chain
    • Protein or peptide: 2F1 Fab light chain
KeywordsE3 Ubiquitin Ligase / Fab / Epitope / Ubiquitination Inhibition / LIGASE / LIGASE-IMMUNE SYSTEM complex
Function / homology
Function and homology information


positive regulation of chaperone-mediated protein complex assembly / regulation of glucocorticoid metabolic process / negative regulation of vascular associated smooth muscle contraction / negative regulation of peroxisome proliferator activated receptor signaling pathway / ubiquitin conjugating enzyme complex / positive regulation of ERAD pathway / positive regulation of mitophagy / positive regulation of smooth muscle cell apoptotic process / ERBB2 signaling pathway / negative regulation of cardiac muscle hypertrophy ...positive regulation of chaperone-mediated protein complex assembly / regulation of glucocorticoid metabolic process / negative regulation of vascular associated smooth muscle contraction / negative regulation of peroxisome proliferator activated receptor signaling pathway / ubiquitin conjugating enzyme complex / positive regulation of ERAD pathway / positive regulation of mitophagy / positive regulation of smooth muscle cell apoptotic process / ERBB2 signaling pathway / negative regulation of cardiac muscle hypertrophy / nuclear inclusion body / misfolded protein binding / cellular response to misfolded protein / protein folding chaperone complex / RIPK1-mediated regulated necrosis / ubiquitin-ubiquitin ligase activity / SMAD binding / protein quality control for misfolded or incompletely synthesized proteins / negative regulation of smooth muscle cell apoptotic process / TPR domain binding / R-SMAD binding / positive regulation of proteolysis / protein K63-linked ubiquitination / protein monoubiquitination / ubiquitin ligase complex / endoplasmic reticulum unfolded protein response / protein autoubiquitination / ERAD pathway / heat shock protein binding / Hsp70 protein binding / Downregulation of TGF-beta receptor signaling / positive regulation of protein ubiquitination / response to ischemia / Regulation of TNFR1 signaling / Hsp90 protein binding / negative regulation of transforming growth factor beta receptor signaling pathway / G protein-coupled receptor binding / regulation of protein stability / RING-type E3 ubiquitin transferase / Regulation of necroptotic cell death / tau protein binding / Z disc / kinase binding / Downregulation of ERBB2 signaling / Regulation of PTEN stability and activity / protein polyubiquitination / ubiquitin-protein transferase activity / Regulation of RUNX2 expression and activity / ubiquitin protein ligase activity / MAPK cascade / Antigen processing: Ubiquitination & Proteasome degradation / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / protein-folding chaperone binding / cellular response to heat / ubiquitin-dependent protein catabolic process / protein-macromolecule adaptor activity / cellular response to hypoxia / proteasome-mediated ubiquitin-dependent protein catabolic process / protein stabilization / protein ubiquitination / DNA repair / ubiquitin protein ligase binding / enzyme binding / endoplasmic reticulum / protein homodimerization activity / mitochondrion / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
CHIP, N-terminal tetratricopeptide repeat domain / CHIP/LubX , U box domain / CHIP N-terminal tetratricopeptide repeat domain / Anaphase-promoting complex, cyclosome, subunit 3 / U-box domain / U-box domain profile. / Modified RING finger domain / U-box domain / TPR repeat region circular profile. / TPR repeat profile. ...CHIP, N-terminal tetratricopeptide repeat domain / CHIP/LubX , U box domain / CHIP N-terminal tetratricopeptide repeat domain / Anaphase-promoting complex, cyclosome, subunit 3 / U-box domain / U-box domain profile. / Modified RING finger domain / U-box domain / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
E3 ubiquitin-protein ligase CHIP
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.02 Å
AuthorsUnnikrishnan A / Southworth D
Funding support1 items
OrganizationGrant numberCountry
Other private
CitationJournal: J Biol Chem / Year: 2025
Title: Recombinant antibodies inhibit enzymatic activity of the E3 ubiquitin ligase CHIP via multiple mechanisms.
Authors: Dong Hee Chung / Emily J Connelly / Aparna Unnikrishnan / Shih-Wei Chuo / Kristin Wucherer / Cory M Nadel / Jason E Gestwicki / Daniel R Southworth / Charles S Craik /
Abstract: Carboxyl-terminus of Hsp70-Interacting Protein (CHIP) is an E3 ubiquitin ligase that marks misfolded substrates for degradation. Hyper-activation of CHIP has been implicated in multiple diseases, ...Carboxyl-terminus of Hsp70-Interacting Protein (CHIP) is an E3 ubiquitin ligase that marks misfolded substrates for degradation. Hyper-activation of CHIP has been implicated in multiple diseases, including cystic fibrosis and cancer, suggesting that it may be a potential drug target. However, there are few tools available for exploring this possibility. Moreover, the best ways of inhibiting CHIP's function are not obvious, as this complex protein is composed of a tetratricopeptide repeat (TPR) domain, a U-box domain, and a coiled-coil domain that mediates homodimerization. To probe the structure and function of CHIP, we report an antibody panning campaign that yielded six recombinant Fabs with affinity for CHIP. Interestingly, these antibodies varied in their binding site(s) and impact on CHIP function, such as inhibiting TPR interactions, autoubiquitination, and/or substrate ubiquitination. Of particular interest, antibody 2F1 nearly eliminated substrate binding (IC = 2.7 μM) and limited ubiquitination and autoubiquitination. Cryo-electron microscopy of the 2F1:CHIP complex revealed a 2:1 binding mode (Fab:CHIP dimer), with 2F1 bound to the U-box domain and simultaneously displacing the TPR domain. Together, these studies provide insight into ways of inhibiting CHIP's activity and provide a series of new probes for exploring the function of this important E3 ubiquitin ligase.
History
DepositionSep 26, 2024-
Header (metadata) releaseMar 12, 2025-
Map releaseMar 12, 2025-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47134.png

Downloads & links

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Map

FileDownload / File: emd_47134.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 420 pix.
= 350.28 Å		0.83 Å/pix.
x 420 pix.
= 350.28 Å		0.83 Å/pix.
x 420 pix.
= 350.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.8784243 - 1.8118633
Average (Standard dev.)-0.00055433024 (±0.035909444)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 350.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map

Fileemd_47134_half_map_1.map
AnnotationHalf Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map

Fileemd_47134_half_map_2.map
AnnotationHalf Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CHIP U-box dimer bound to Fab 2F1

EntireName: CHIP U-box dimer bound to Fab 2F1
Components
  • Complex: CHIP U-box dimer bound to Fab 2F1
    • Protein or peptide: E3 ubiquitin-protein ligase CHIP
    • Protein or peptide: 2F1 Fab heavy chain
    • Protein or peptide: 2F1 Fab light chain

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Supramolecule #1: CHIP U-box dimer bound to Fab 2F1

SupramoleculeName: CHIP U-box dimer bound to Fab 2F1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: E3 ubiquitin-protein ligase CHIP

MacromoleculeName: E3 ubiquitin-protein ligase CHIP / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.626804 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
DIPDYLCGKI SFELMREPCI TPSGITYDRK DIEEHLQRVG HFDPVTRSPL TQEQLIPNLA MKEVIDAFIS ENGWV

UniProtKB: E3 ubiquitin-protein ligase CHIP

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Macromolecule #2: 2F1 Fab heavy chain

MacromoleculeName: 2F1 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.43223 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQP PGKGLEWIGE INHSGSTNYN PSLKSRVTIS VDTSKNQFSL KLSSVTAAD TAVYYCARGG VGRVRGDSMD VWGQGTTVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT ...String:
QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQP PGKGLEWIGE INHSGSTNYN PSLKSRVTIS VDTSKNQFSL KLSSVTAAD TAVYYCARGG VGRVRGDSMD VWGQGTTVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPK

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Macromolecule #3: 2F1 Fab light chain

MacromoleculeName: 2F1 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.929486 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: PVLTQPPSVS KALRQTATLT CTGDSNNVGN RGAAWFLLHQ GHPPKLLAYR NNDRPSGISE RLSASRSGNT ASLTITGLQP EDEADYYCS AWDSSLRVQV FGGGTKLTVL GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK A GVETTTPS ...String:
PVLTQPPSVS KALRQTATLT CTGDSNNVGN RGAAWFLLHQ GHPPKLLAYR NNDRPSGISE RLSASRSGNT ASLTITGLQP EDEADYYCS AWDSSLRVQV FGGGTKLTVL GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK A GVETTTPS KQSNNKYAAS SYLSLTPEQW KSHKSYSCQV THEGSTVEKT VAPTECS

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Details: Wait time 10 sec, blot time 3 sec, blot force 0, Graphene Oxide coated grids.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio reconstruction from cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.02 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 16310
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD

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