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- PDB-7qun: CryoEM structure of mammalian AAP in complex with Meropenem -

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Basic information

Entry
Database: PDB / ID: 7qun
TitleCryoEM structure of mammalian AAP in complex with Meropenem
ComponentsAcylamino-acid-releasing enzymeAcylaminoacyl-peptidase
KeywordsHYDROLASE / inhibitor / complex / acylaminoacyl-peptidase / meropenem / carbapenem / acylpeptide hydrolase
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
Chem-DWZ / Acylamino-acid-releasing enzyme
Similarity search - Component
Biological speciesSus scrofa domesticus (domestic pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsKiss-Szeman, A.J. / Harmat, V. / Straner, P. / Jakli, I. / Menyhard, K.D. / Masiulis, S. / 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: A carbapenem antibiotic inhibiting a mammalian serine protease: structure of the acylaminoacyl peptidase-meropenem complex.
Authors: Anna J Kiss-Szemán / Luca Takács / Zoltán Orgován / Pál Stráner / Imre Jákli / Gitta Schlosser / Simonas Masiulis / Veronika Harmat / Dóra K Menyhárd / András Perczel /
Abstract: The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem ...The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem antibiotic. AAP is a modulator of the ubiquitin-proteasome degradation system and the site of a drug-drug interaction between the widely used antipsychotic, valproate and carbapenems. The active form of pAAP - a toroidal tetramer - binds four meropenem molecules covalently linked to the catalytic Ser587 of the serine-protease triad, in an acyl-enzyme state. AAP is hindered from fully processing the antibiotic by the displacement and protonation of His707 of the catalytic triad. We show that AAP is made susceptible to the association by its unusually sheltered active pockets and flexible catalytic triads, while the carbapenems possess sufficiently small substituents on their β-lactam rings to fit into the shallow substrate-specificity pocket of the enzyme.
History
DepositionJan 18, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 1, 2023Group: 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

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)326,8398
Polymers325,2984
Non-polymers1,5424
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18200 Å2
ΔGint-99 kcal/mol
Surface area99570 Å2
MethodPISA

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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
#2: Chemical
ChemComp-DWZ / (2S,3R,4S)-4-{[(3S,5S)-5-(dimethylcarbamoyl)pyrrolidin-3-yl]sulfanyl}-2-[(2S,3R)-3-hydroxy-1-oxobutan-2-yl]-3-methyl-3,4-dihydro-2H-pyrrole-5-carboxylic acid / Meropenem, bound form / Meropenem


Mass: 385.478 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C17H27N3O5S / Feature type: SUBJECT OF INVESTIGATION / Comment: antibiotic*YM
Has ligand of interestY

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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 aclyaminoacyl-peptidase / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Sus scrofa domesticus (domestic pig)
Buffer solutionpH: 7.5
Buffer componentConc.: 10 mM / Name: TRIS / Formula: C4H11NO3
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Covalent complex with carbapenem type antibiotic Meropenem
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 5.93 sec. / Electron dose: 41 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 11625

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
4cryoSPARC3.2.0CTF correction
10cryoSPARC3.2.0initial Euler assignment
11cryoSPARC3.2.0final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 654863 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221669
ELECTRON MICROSCOPYf_angle_d0.48129561
ELECTRON MICROSCOPYf_dihedral_angle_d4.3882988
ELECTRON MICROSCOPYf_chiral_restr0.0433312
ELECTRON MICROSCOPYf_plane_restr0.0043785

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