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- PDB-7px8: CryoEM structure of mammalian acylaminoacyl-peptidase -

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Basic information

Entry
Database: PDB / ID: 7px8
TitleCryoEM structure of mammalian acylaminoacyl-peptidase
ComponentsAcylamino-acid-releasing enzymeAcylaminoacyl-peptidase
KeywordsHYDROLASE / acylaminoacyl-peptidase / tetramer / aclypeptide-hydrolase / oxidized protein hydrolase / serine-protease
Function / homology
Function and homology information


acylaminoacyl-peptidase / serine-type endopeptidase activity / proteolysis / cytoplasm
Similarity search - Function
Acylamino-acid-releasing enzyme, N-terminal domain / Acylamino-acid-releasing enzyme, N-terminal domain / Prolyl endopeptidase family serine active site. / Peptidase S9, serine active site / Peptidase S9, prolyl oligopeptidase, catalytic domain / Prolyl oligopeptidase family / Six-bladed beta-propeller, TolB-like / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Acylamino-acid-releasing enzyme
Similarity search - Component
Biological speciesSus scrofa domesticus (domestic pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsKiss-Szeman, A.J. / Harmat, V. / Menyhard, D.K. / Straner, P. / Jakli, I. / Hosogi, N. / Perczel, A.
Funding support Hungary, 5items
OrganizationGrant numberCountry
European Regional Development FundVEKOP-2.3.2-16-2017-00014 Hungary
European Regional Development FundVEKOP-2.3.3-15-2017-00018 Hungary
Ministry of Human Capacities2018-1.2.1-NKP-2018-00005 Hungary
National Research Development and Innovation Office (NKFIH)Thematic Excellence Program 2019, Synth+ Hungary
National Research Development and Innovation Office (NKFIH)K116305 Hungary
CitationJournal: Chem Sci / Year: 2022
Title: Cryo-EM structure of acylpeptide hydrolase reveals substrate selection by multimerization and a multi-state serine-protease triad.
Authors: Anna J Kiss-Szemán / Pál Stráner / Imre Jákli / Naoki Hosogi / Veronika Harmat / Dóra K Menyhárd / András Perczel /
Abstract: The first structure of tetrameric mammalian acylaminoacyl peptidase, an enzyme that functions as an upstream regulator of the proteasome through the removal of terminal -acetylated residues from its ...The first structure of tetrameric mammalian acylaminoacyl peptidase, an enzyme that functions as an upstream regulator of the proteasome through the removal of terminal -acetylated residues from its protein substrates, was determined by cryo-EM and further elucidated by MD simulations. Self-association results in a toroid-shaped quaternary structure, guided by an amyloidogenic β-edge and unique inserts. With a Pro introduced into its central β-sheet, sufficient conformational freedom is awarded to the segment containing the catalytic Ser587 that the serine protease catalytic triad alternates between active and latent states. Active site flexibility suggests that the dual function of catalysis and substrate selection are fulfilled by a novel mechanism: substrate entrance is regulated by flexible loops creating a double-gated channel system, while binding of the substrate to the active site is required for stabilization of the catalytic apparatus - as a second filter before hydrolysis. The structure not only underlines that within the family of S9 proteases homo-multimerization acts as a crucial tool for substrate selection, but it will also allow drug design targeting of the ubiquitin-proteasome system.
History
DepositionOct 8, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 25, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 1, 2022Group: Data collection / Category: em_imaging
Item: _em_imaging.nominal_defocus_max / _em_imaging.nominal_defocus_min
Revision 1.2Jul 20, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Acylamino-acid-releasing enzyme
B: Acylamino-acid-releasing enzyme
C: Acylamino-acid-releasing enzyme
D: Acylamino-acid-releasing enzyme


Theoretical massNumber of molelcules
Total (without water)325,2984
Polymers325,2984
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18190 Å2
ΔGint-105 kcal/mol
Surface area102860 Å2

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Components

#1: Protein
Acylamino-acid-releasing enzyme / Acylaminoacyl-peptidase


Mass: 81324.391 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / References: UniProt: P19205

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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: Homotetramer of acylaminoacyl-peptidase / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / Organ: liver
Buffer solutionpH: 7.5
Buffer componentConc.: 10 mM / Name: TRIS / Formula: C4H11NO3
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL
Image recordingAverage exposure time: 4 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1157
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
2JADASimage acquisition
4CTFFINDCTF correction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50604 / Symmetry type: POINT

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