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- PDB-6b3q: Cryo-EM structure of human insulin degrading enzyme in complex wi... -

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Basic information

Entry
Database: PDB / ID: 6b3q
TitleCryo-EM structure of human insulin degrading enzyme in complex with insulin
Components
  • Insulin-degrading enzyme
  • Insulin
KeywordsHYDROLASE/HORMONE / IDE / insulin degrading enzyme / amyloid beta / HYDROLASE-HORMONE complex
Function / homologyMiddle or third domain of peptidase_M16 / Insulin family signature. / Insulin-like / Insulin family / Peptidase M16, C-terminal / Insulin, conserved site / Peptidase M16, middle/third domain / Insulin-like superfamily / Insulin/IGF/Relaxin family / Insulinase (Peptidase family M16) ...Middle or third domain of peptidase_M16 / Insulin family signature. / Insulin-like / Insulin family / Peptidase M16, C-terminal / Insulin, conserved site / Peptidase M16, middle/third domain / Insulin-like superfamily / Insulin/IGF/Relaxin family / Insulinase (Peptidase family M16) / Peptidase M16 inactive domain / Insulin / Peptidase M16, zinc-binding site / Insulinase family, zinc-binding region signature. / Regulation of gene expression in beta cells / Metalloenzyme, LuxS/M16 peptidase-like / Insulin processing / Synthesis, secretion, and deacylation of Ghrelin / Regulation of insulin secretion / Ub-specific processing proteases / COPI-mediated anterograde transport / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / IRS activation / Signal attenuation / Insulin receptor signalling cascade / Signaling by Insulin receptor / Insulin receptor recycling / Peroxisomal protein import / Amyloid fiber formation / Peptidase M16, N-terminal / insulysin / beta-endorphin binding / bradykinin catabolic process / insulin catabolic process / insulin metabolic process / ubiquitin recycling / ubiquitin-dependent protein binding / hormone catabolic process / cytosolic proteasome complex / insulin binding / amyloid-beta clearance / alpha-beta T cell activation / negative regulation of glycogen catabolic process / negative regulation of fatty acid metabolic process / negative regulation of feeding behavior / negative regulation of NAD(P)H oxidase activity / determination of adult lifespan / regulation of cellular amino acid metabolic process / regulation of transmembrane transporter activity / positive regulation of respiratory burst / regulation of protein secretion / negative regulation of respiratory burst involved in inflammatory response / peroxisomal matrix / negative regulation of protein oligomerization / positive regulation of peptide hormone secretion / positive regulation of dendritic spine maintenance / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / positive regulation of lipid biosynthetic process / negative regulation of blood vessel diameter / negative regulation of gluconeogenesis / negative regulation of protein secretion / positive regulation of protein oligomerization / negative regulation of reactive oxygen species biosynthetic process / cognition / negative regulation of lipid catabolic process / positive regulation of cellular protein metabolic process / fatty acid homeostasis / regulation of protein localization to plasma membrane / positive regulation of glycolytic process / positive regulation of insulin receptor signaling pathway / positive regulation of glycogen biosynthetic process / positive regulation of nitric oxide mediated signal transduction / transport vesicle / endoplasmic reticulum-Golgi intermediate compartment membrane / regulation of synaptic plasticity / endosome lumen / positive regulation of brown fat cell differentiation / insulin-like growth factor receptor binding / neuron projection maintenance / positive regulation of protein autophosphorylation / amyloid-beta metabolic process / positive regulation of glucose import in response to insulin stimulus / protein heterooligomerization / positive regulation of cell differentiation / negative regulation of acute inflammatory response / regulation of protein localization / positive regulation of cytokine secretion / proteolysis involved in cellular protein catabolic process / positive regulation of mitotic nuclear division / protein targeting to peroxisome / positive regulation of protein catabolic process / positive regulation of long-term synaptic potentiation / peptide binding / positive regulation of glucose import / activation of protein kinase B activity / negative regulation of protein catabolic process / positive regulation of nitric-oxide synthase activity / negative regulation of proteolysis / insulin receptor binding / insulin receptor signaling pathway
Function and homology information
Specimen sourceHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.7 Å resolution
AuthorsLiang, W.G. / Zhang, Z. / Bailey, L.J. / Kossiakoff, A.A. / Tan, Y.Z. / Wei, H. / Carragher, B. / Potter, S.C. / Tang, W.J.
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-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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Sep 22, 2017 / Release: Nov 22, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 22, 2017Structure modelrepositoryInitial release
1.1Dec 6, 2017Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization
1.2Apr 11, 2018Structure modelData collection / Database referencescitation / citation_author_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
1.3Jul 18, 2018Structure modelData collectionem_software_em_software.name

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Structure visualization

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  • Deposited structure unit
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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)253,1034
Polyers253,1034
Non-polymers00
Water0
1
A: Insulin-degrading enzyme
a: Insulin


Theoretical massNumber of molelcules
Total (without water)126,5512
Polyers126,5512
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
B: Insulin-degrading enzyme
b: Insulin


Theoretical massNumber of molelcules
Total (without water)126,5512
Polyers126,5512
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 114561.562 Da / Num. of mol.: 2 / Fragment: residues 42-1019 / Source: (gene. exp.) Homo sapiens (human) / Gene: IDE / Production host: Escherichia coli (E. coli) / References: UniProt: P14735, insulysin
#2: Protein/peptide Insulin /


Mass: 11989.862 Da / Num. of mol.: 2 / Source: (gene. exp.) Homo sapiens (human) / Gene: INS / Production host: Escherichia coli (E. coli) / References: UniProt: P01308

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Insulin degrading enzyme/Insulin / Type: COMPLEX
Details: Cryo-EM structure of human insulin degrading enzyme in complex with insulin
Entity ID: 1,2 / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer ID
120 mmol/LHEPESC8H18N2O4S1
2300 mmol/LSodium chlorideNaCl1
320 mmol/LEDTAC10H16N2O81
SpecimenConc.: 0.3 mg/ml / Details: The sample was monodisperse / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 / Grid type: homemade nanowire grid
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 298 kelvins
Details: The cryo grids were made using Spotiton and homemade plunger

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 / Calibrated magnification: 46598 / Nominal defocus max: 2200 nm / Nominal defocus min: 940 nm / Cs: 2.7 mm / C2 aperture diameter: 70 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 kelvins / Temperature (min): 70 kelvins / 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) / Number of grids imaged: 3 / Number of real images: 3085
Image scansSampling size: 5 microns / 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_cu7.5_x86_64CTF correction
7CootCoot 0.8.9_premodel fitting
8UCSF ChimeraChimera 1.11.2model fitting
10RELION2.0initial Euler assignment
11RELION2.0final Euler assignment
12RELION2.0classification
13RELION2.13D reconstruction
20PHENIXPhenix-1.12-2829model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 762283
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 116122 / Algorithm: FOURIER SPACE / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingOverall b value: 92 / Ref protocol: FLEXIBLE FIT / Ref space: REAL
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00516115
ELECTRON MICROSCOPYf_angle_d1.04121795
ELECTRON MICROSCOPYf_dihedral_angle_d9.0219745
ELECTRON MICROSCOPYf_chiral_restr0.0592352
ELECTRON MICROSCOPYf_plane_restr0.0082815

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