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- PDB-6b7z: Cryo-EM structure of human insulin degrading enzyme in complex wi... -

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Basic information

Entry
Database: PDB / ID: 6b7z
TitleCryo-EM structure of human insulin degrading enzyme in complex with FAB H11 heavy chain and FAB H11 light chain
Components
  • FAB H11 heavy chain
  • FAB H11 light chain
  • Insulin-degrading enzyme
KeywordsHYDROLASE/IMMUNE SYSTEM / IDE / insulin degrading enzyme / amyloid beta / BIOSYNTHETIC PROTEIN / HYDROLASE-IMMUNE SYSTEM 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 / regulation of aerobic respiration / peptide catabolic process ...insulysin / ubiquitin recycling / insulin catabolic process / insulin metabolic process / amyloid-beta clearance by cellular catabolic process / hormone catabolic process / bradykinin catabolic process / insulin binding / regulation of aerobic respiration / peptide catabolic process / immunoglobulin complex / amyloid-beta clearance / peroxisomal matrix / amyloid-beta metabolic process / Insulin receptor recycling / proteolysis involved in protein catabolic process / Peroxisomal protein import / peptide binding / protein catabolic process / antigen processing and presentation of endogenous peptide antigen via MHC class I / metalloendopeptidase activity / positive regulation of protein catabolic process / peroxisome / positive regulation of protein binding / insulin receptor signaling pathway / virus receptor activity / basolateral plasma membrane / endopeptidase activity / adaptive immune response / Ub-specific processing proteases / immune response / external side of plasma membrane / cell surface / protein homodimerization activity / mitochondrion / proteolysis / extracellular space / extracellular exosome / zinc ion binding / extracellular region / ATP binding / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Peptidase M16, middle/third domain / Middle or third domain of peptidase_M16 / PQQ synthase PqqF-like, C-terminal lobe domain 4 / : / 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 / PQQ synthase PqqF-like, C-terminal lobe domain 4 / : / 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 / : / : / 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 / Immunoglobulin-like fold
Similarity search - Domain/homology
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: 6.5 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 5, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 10, 2018Provider: 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
Revision 1.5Oct 9, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-7066
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)316,0246
Polymers316,0246
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12940 Å2
ΔGint-65 kcal/mol
Surface area127940 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 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 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
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 degrading enzyme / Type: COMPLEX
Details: Cryo-EM structure of human Apo insulin degrading enzyme
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 chlorideNaCl1
320 mmol/LEDTAC10H16N2O81
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse
Specimen supportDetails: The grids are homemade lacey gold nanowire grids / Grid material: GOLD / 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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: The image was collected at 20-50 degree tilt
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 recording

Imaging-ID: 1 / Average exposure time: 10 sec. / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

IDElectron dose (e/Å2)Num. of real images
17.9509
26.8620
Image scansSampling size: 5 µm / Width: 3710 / Height: 3820 / 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
10PHENIXPhenix-1.12-2829model refinement
11RELION2initial Euler assignment
12RELION2final Euler assignment
13RELION2classification
14RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 762283
3D reconstructionResolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16944 / 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_d0.00822447
ELECTRON MICROSCOPYf_angle_d1.33930423
ELECTRON MICROSCOPYf_dihedral_angle_d8.77413510
ELECTRON MICROSCOPYf_chiral_restr0.073329
ELECTRON MICROSCOPYf_plane_restr0.0083913

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