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- PDB-6bfc: Cryo-EM structure of human insulin degrading enzyme in complex wi... -

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Basic information

Entry
Database: PDB / ID: 6bfc
TitleCryo-EM structure of human insulin degrading enzyme in complex with insulin
Components
  • Insulin
  • Insulin-degrading enzyme
KeywordsHYDROLASE/HORMONE / IDE / amyloid beta / HORMONE / HYDROLASE-HORMONE complex
Function / homology
Function and homology information


insulysin / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process / bradykinin catabolic process / insulin binding / negative regulation of NAD(P)H oxidase activity / regulation of aerobic respiration ...insulysin / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process / bradykinin catabolic process / insulin binding / negative regulation of NAD(P)H oxidase activity / regulation of aerobic respiration / negative regulation of glycogen catabolic process / positive regulation of nitric oxide mediated signal transduction / negative regulation of fatty acid metabolic process / Signaling by Insulin receptor / negative regulation of feeding behavior / peptide catabolic process / IRS activation / Insulin processing / regulation of protein secretion / positive regulation of peptide hormone secretion / positive regulation of respiratory burst / amyloid-beta clearance / Regulation of gene expression in beta cells / negative regulation of acute inflammatory response / peroxisomal matrix / positive regulation of protein autophosphorylation / alpha-beta T cell activation / positive regulation of dendritic spine maintenance / Synthesis, secretion, and deacylation of Ghrelin / negative regulation of respiratory burst involved in inflammatory response / amyloid-beta metabolic process / negative regulation of protein secretion / negative regulation of gluconeogenesis / positive regulation of glycogen biosynthetic process / Signal attenuation / fatty acid homeostasis / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / positive regulation of insulin receptor signaling pathway / negative regulation of lipid catabolic process / regulation of protein localization to plasma membrane / positive regulation of lipid biosynthetic process / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / activation of protein kinase B activity / COPI-mediated anterograde transport / transport vesicle / nitric oxide-cGMP-mediated signaling / negative regulation of reactive oxygen species biosynthetic process / Insulin receptor recycling / insulin-like growth factor receptor binding / positive regulation of brown fat cell differentiation / NPAS4 regulates expression of target genes / neuron projection maintenance / endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of nitric-oxide synthase activity / positive regulation of mitotic nuclear division / Insulin receptor signalling cascade / regulation of transmembrane transporter activity / peptide binding / proteolysis involved in protein catabolic process / positive regulation of glycolytic process / positive regulation of long-term synaptic potentiation / positive regulation of cytokine production / endosome lumen / acute-phase response / positive regulation of D-glucose import / positive regulation of protein secretion / positive regulation of cell differentiation / Regulation of insulin secretion / insulin receptor binding / Peroxisomal protein import / wound healing / protein catabolic process / negative regulation of protein catabolic process / antigen processing and presentation of endogenous peptide antigen via MHC class I / hormone activity / regulation of synaptic plasticity / positive regulation of neuron projection development / metalloendopeptidase activity / positive regulation of protein localization to nucleus / Golgi lumen / cognition / glucose metabolic process / positive regulation of protein catabolic process / positive regulation of protein binding / vasodilation / peroxisome / insulin receptor signaling pathway / glucose homeostasis / cell-cell signaling / regulation of protein localization / virus receptor activity / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / protease binding / positive regulation of cell growth / basolateral plasma membrane / secretory granule lumen / endopeptidase activity / positive regulation of canonical NF-kappaB signal transduction / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of MAPK cascade
Similarity search - Function
Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / : / PQQ synthase PqqF-like, C-terminal lobe domain 4 / : / Cytochrome Bc1 Complex; Chain A, domain 1 / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Peptidase M16, C-terminal ...Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / : / PQQ synthase PqqF-like, C-terminal lobe domain 4 / : / Cytochrome Bc1 Complex; Chain A, domain 1 / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Peptidase M16, C-terminal / Peptidase M16 inactive domain / Peptidase M16, N-terminal / Insulinase (Peptidase family M16) / Metalloenzyme, LuxS/M16 peptidase-like / Insulin / Insulin family / Insulin-like / Insulin/IGF/Relaxin family / Insulin / insulin-like growth factor / relaxin family. / Insulin, conserved site / Insulin family signature. / Insulin-like superfamily / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Insulin / Insulin-degrading enzyme
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsLiang, W.G. / Zhang, Z. / Bailey, L.J. / Kossiakoff, A.A. / Tan, Y.Z. / Wei, H. / Carragher, B. / Potter, S.C. / Tang, W.J.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM81539 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM121964 United States
Simons Foundation349247 United States
CitationJournal: Elife / Year: 2018
Title: Ensemble cryoEM elucidates the mechanism of insulin capture and degradation by human insulin degrading enzyme.
Authors: Zhening Zhang / Wenguang G Liang / Lucas J Bailey / Yong Zi Tan / Hui Wei / Andrew Wang / Mara Farcasanu / Virgil A Woods / Lauren A McCord / David Lee / Weifeng Shang / Rebecca Deprez- ...Authors: Zhening Zhang / Wenguang G Liang / Lucas J Bailey / Yong Zi Tan / Hui Wei / Andrew Wang / Mara Farcasanu / Virgil A Woods / Lauren A McCord / David Lee / Weifeng Shang / Rebecca Deprez-Poulain / Benoit Deprez / David R Liu / Akiko Koide / Shohei Koide / Anthony A Kossiakoff / Sheng Li / Bridget Carragher / Clinton S Potter / Wei-Jen Tang /
Abstract: Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes ...Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.
History
DepositionOct 26, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 27, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2018Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Apr 11, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization
Revision 1.4Apr 28, 2021Group: Derived calculations
Category: pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop
Revision 1.5Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Insulin-degrading enzyme
B: Insulin-degrading enzyme
a: Insulin
b: Insulin


Theoretical massNumber of molelcules
Total (without water)247,7134
Polymers247,7134
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8250 Å2
ΔGint-29 kcal/mol
Surface area79430 Å2

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Components

#1: Protein Insulin-degrading enzyme / Abeta-degrading protease / Insulin protease / Insulinase / Insulysin


Mass: 111866.484 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IDE / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P14735, insulysin
#2: Protein Insulin


Mass: 11989.862 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P01308
Has protein modificationY

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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: Insulin bound insulin degrading enzyme / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mmol/lHEPEsC8H18N2O4S1
2300 mmol/lSodium ChlorideNaCl1
320 mmol/lEDTAC10H16N2O81
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Homemade
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 293 K / Details: Grids made using Spotiton

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Calibrated magnification: 46598 X / Nominal defocus max: 2200 nm / Nominal defocus min: 940 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K / Residual tilt: 10 mradians
Image recordingAverage exposure time: 10 sec. / Electron dose: 71.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 3085
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
1DoG Pickerparticle selection
2Leginon3.3image acquisition
4GctfGctf_v0.50_sm_30_cu7.5X86_64CTF correction
7Coot0.89_Premodel fitting
8UCSF Chimera1.11.2model fitting
10RELION2initial Euler assignment
12RELION2classification
13RELION2.13D reconstruction
14PHENIX1.02-2829model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 762283
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 148392 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 111.9 / Protocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00616160
ELECTRON MICROSCOPYf_angle_d1.04921857
ELECTRON MICROSCOPYf_dihedral_angle_d9.0169765
ELECTRON MICROSCOPYf_chiral_restr0.0592362
ELECTRON MICROSCOPYf_plane_restr0.0082822

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