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- PDB-6b70: Cryo-EM structure of human insulin degrading enzyme in complex wi... -

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Basic information

Entry
Database: PDB / ID: 6b70
TitleCryo-EM structure of human insulin degrading enzyme in complex with FAB H11-E heavy chain, FAB H11-E light chain and insulin
Components
  • FAB H11-E heavy chain
  • FAB H11-E light chain
  • Insulin-degrading enzyme
  • Insulin
KeywordsHYDROLASE/IMMUNE SYSTEM/HORMONE / IDE / insulin degrading enzyme / amyloid beta / BIOSYNTHETIC PROTEIN / HYDROLASE-IMMUNE SYSTEM-HORMONE complex
Function / homologyInsulin-like superfamily / Insulin-like / Peptidase M16 inactive domain / Insulinase (Peptidase family M16) / Insulin/IGF/Relaxin family / Immunoglobulin-like domain superfamily / Peptidase M16, middle/third domain / Insulin, conserved site / Insulin family / Immunoglobulin-like fold ...Insulin-like superfamily / Insulin-like / Peptidase M16 inactive domain / Insulinase (Peptidase family M16) / Insulin/IGF/Relaxin family / Immunoglobulin-like domain superfamily / Peptidase M16, middle/third domain / Insulin, conserved site / Insulin family / Immunoglobulin-like fold / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin V-set domain / Peptidase M16, N-terminal / Metalloenzyme, LuxS/M16 peptidase-like / Peptidase M16, C-terminal / Immunoglobulin-like domain / Insulin / Immunoglobulin subtype / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Immunoglobulin V-set domain / Middle or third domain of peptidase_M16 / COPI-mediated anterograde transport / Amyloid fiber formation / Peroxisomal protein import / Insulin receptor recycling / Signaling by Insulin receptor / Insulin receptor signalling cascade / Signal attenuation / IRS activation / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / Ub-specific processing proteases / Insulinase family, zinc-binding region signature. / Regulation of insulin secretion / Synthesis, secretion, and deacylation of Ghrelin / Insulin processing / Regulation of gene expression in beta cells / Ig-like domain profile. / Immunoglobulins and major histocompatibility complex proteins signature. / Insulin family signature. / Peptidase M16, zinc-binding site / beta-endorphin binding / insulysin / bradykinin catabolic process / insulin catabolic process / ubiquitin recycling / insulin metabolic process / ubiquitin-dependent protein binding / hormone catabolic process / cytosolic proteasome complex / insulin binding / amyloid-beta clearance / negative regulation of glycogen catabolic process / alpha-beta T cell activation / 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 / regulation of protein secretion / positive regulation of respiratory burst / peroxisomal matrix / negative regulation of respiratory burst involved in inflammatory response / 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 glycogen biosynthetic process / positive regulation of insulin receptor signaling pathway / 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 / protein heterooligomerization / amyloid-beta metabolic process / positive regulation of glucose import in response to insulin stimulus / positive regulation of cell differentiation / proteolysis involved in cellular protein catabolic process / negative regulation of acute inflammatory response / regulation of protein localization / positive regulation of cytokine secretion / positive regulation of mitotic nuclear division
Function and homology information
Specimen sourceHomo sapiens (human)
Mus musculus (house mouse)
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: Oct 3, 2017 / Release: Dec 27, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 27, 2017Structure modelrepositoryInitial release
1.1Jan 17, 2018Structure 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

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Structure visualization

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Assembly

Deposited unit
A: Insulin-degrading enzyme
B: Insulin-degrading enzyme
C: FAB H11-E heavy chain
D: FAB H11-E light chain
E: FAB H11-E heavy chain
F: FAB H11-E light chain
a: Insulin
c: Insulin


Theoretical massNumber of molelcules
Total (without water)340,0038
Polyers340,0038
Non-polymers00
Water0
1
A: Insulin-degrading enzyme
C: FAB H11-E heavy chain
D: FAB H11-E light chain
a: Insulin


Theoretical massNumber of molelcules
Total (without water)170,0024
Polyers170,0024
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
B: Insulin-degrading enzyme
E: FAB H11-E heavy chain
F: FAB H11-E light chain
c: Insulin


Theoretical massNumber of molelcules
Total (without water)170,0024
Polyers170,0024
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: 111866.484 Da / Num. of mol.: 2 / Fragment: residues 46-1011 / Source: (gene. exp.) Homo sapiens (human) / Gene: IDE / Production host: Escherichia coli (E. coli) / References: UniProt: P14735, insulysin
#2: Protein/peptide FAB H11-E heavy chain


Mass: 23057.748 Da / Num. of mol.: 2
Source: (gene. exp.) Mus musculus (house mouse), (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli (E. coli) / References: UniProt: P0DOX5
#3: Protein/peptide FAB H11-E light chain


Mass: 23087.609 Da / Num. of mol.: 2
Source: (gene. exp.) Mus musculus (house mouse), (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli (E. coli) / References: UniProt: Q6GMX0
#4: 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: 1,2,3,4 / 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
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
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_d0.01122760
ELECTRON MICROSCOPYf_angle_d1.28530851
ELECTRON MICROSCOPYf_dihedral_angle_d9.46613700
ELECTRON MICROSCOPYf_chiral_restr0.0673380
ELECTRON MICROSCOPYf_plane_restr0.0103967

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