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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 / 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 / immunoglobulin complex / ubiquitin-dependent protein binding / insulin binding ...insulysin / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process / bradykinin catabolic process / immunoglobulin complex / ubiquitin-dependent protein binding / insulin binding / negative regulation of NAD(P)H oxidase activity / regulation of aerobic respiration / negative regulation of glycogen catabolic process / regulation of cellular amino acid metabolic process / Signaling by Insulin receptor / peptide catabolic process / IRS activation / negative regulation of fatty acid metabolic process / nitric oxide-cGMP-mediated signaling / Insulin processing / negative regulation of feeding behavior / regulation of protein secretion / amyloid-beta clearance / positive regulation of peptide hormone secretion / Regulation of gene expression in beta cells / peroxisomal matrix / positive regulation of respiratory burst / positive regulation of dendritic spine maintenance / alpha-beta T cell activation / negative regulation of acute inflammatory response / negative regulation of respiratory burst involved in inflammatory response / negative regulation of protein secretion / fatty acid homeostasis / Synthesis, secretion, and deacylation of Ghrelin / positive regulation of glycogen biosynthetic process / positive regulation of lipid biosynthetic process / Signal attenuation / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / negative regulation of gluconeogenesis / regulation of protein localization to plasma membrane / positive regulation of nitric oxide mediated signal transduction / COPI-mediated anterograde transport / negative regulation of lipid catabolic process / amyloid-beta metabolic process / negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway / negative regulation of reactive oxygen species biosynthetic process / positive regulation of insulin receptor signaling pathway / transport vesicle / positive regulation of protein autophosphorylation / insulin-like growth factor receptor binding / Insulin receptor recycling / positive regulation of protein metabolic process / NPAS4 regulates expression of target genes / endoplasmic reticulum-Golgi intermediate compartment membrane / neuron projection maintenance / positive regulation of brown fat cell differentiation / activation of protein kinase B activity / positive regulation of glycolytic process / proteolysis involved in protein catabolic process / Insulin receptor signalling cascade / positive regulation of mitotic nuclear division / Regulation of insulin secretion / positive regulation of nitric-oxide synthase activity / positive regulation of long-term synaptic potentiation / endosome lumen / positive regulation of cytokine production / acute-phase response / regulation of transmembrane transporter activity / positive regulation of protein secretion / positive regulation of cell differentiation / Peroxisomal protein import / peptide binding / positive regulation of glucose import / negative regulation of proteolysis / protein catabolic process / regulation of synaptic plasticity / wound healing / insulin receptor binding / cognition / negative regulation of protein catabolic process / positive regulation of neuron projection development / hormone activity / metalloendopeptidase activity / antigen processing and presentation of endogenous peptide antigen via MHC class I / vasodilation / Golgi lumen / peroxisome / positive regulation of protein localization to nucleus / positive regulation of protein catabolic process / glucose metabolic process / regulation of protein localization / glucose homeostasis / cell-cell signaling / virus receptor activity / insulin receptor signaling pathway / positive regulation of NF-kappaB transcription factor activity / positive regulation of protein binding / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / positive regulation of cell growth / basolateral plasma membrane
Similarity search - Function
Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / 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) ...Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / 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/IGF/Relaxin family / Insulin, conserved site / Insulin family signature. / Insulin-like / Insulin / insulin-like growth factor / relaxin family. / Insulin-like superfamily / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin subtype / Immunoglobulin / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulins / Immunoglobulin-like fold / Immunoglobulin-like / Sandwich / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Insulin / Immunoglobulin gamma-1 heavy chain / Insulin-degrading enzyme / Ig-like domain-containing protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
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 3, 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 1.3Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Apr 28, 2021Group: Derived calculations
Category: pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop

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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
Polymers340,0038
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18280 Å2
ΔGint-87 kcal/mol
Surface area115390 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 / Fragment: residues 46-1011
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IDE / Production host: Escherichia coli (E. coli) / References: UniProt: P14735, insulysin
#2: Antibody FAB H11-E heavy chain


Mass: 23057.748 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse), (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli (E. coli) / References: UniProt: P0DOX5
#3: Antibody FAB H11-E light chain


Mass: 23087.609 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse), (gene. exp.) Homo sapiens (human)
Production host: Escherichia coli (E. coli) / References: UniProt: Q6GMX0
#4: 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) / References: UniProt: P01308

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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 degrading enzyme/Insulin / Type: COMPLEX
Details: Cryo-EM structure of human insulin degrading enzyme in complex with insulin
Entity ID: all / 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 chlorideNaClSodium chloride1
320 mmol/LEDTAEthylenediaminetetraacetic acidC10H16N2O81
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Homemade
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 298 K
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
MicroscopyModel: 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 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_cu7.5_x86_64CTF correction
7CootCoot 0.8.9_premodel fitting
8UCSF Chimerachimera-1.11.2model fitting
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION2.13D reconstruction
20PHENIXPhenix-1.12-2829model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 762283
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 116122 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 92 / Protocol: FLEXIBLE FIT / 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_restr0.013967

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