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- PDB-9hxq: Cryo-EM structure of acylaminoacyl peptidase (AAP) in covalent co... -

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Basic information

Entry
Database: PDB / ID: 9hxq
TitleCryo-EM structure of acylaminoacyl peptidase (AAP) in covalent complex with inhibitor AEBSF
ComponentsAcylamino-acid-releasing enzyme
KeywordsHYDROLASE / inhibitor / covalent / serine-protease / acylpeptide hydrolase / APEH / OPH / acylaminoacyl peptidase / sulphonate
Function / homology
Function and homology information


acylaminoacyl-peptidase / omega peptidase activity / 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
4-(2-AMINOETHYL)BENZENESULFONYL FLUORIDE / Acylamino-acid-releasing enzyme
Similarity search - Component
Biological speciesSus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKiss-Szeman, A.J. / Menyhard, D.K. / Harmat, V. / Perczel, A.
Funding support Hungary, European Union, 3items
OrganizationGrant numberCountry
National Research Development and Innovation Office (NKFIH)2018-1.2.1-NKP-2018-00005 Hungary
European Union (EU)RRF-2.3.1-21-2022-00015European Union
National Research Development and Innovation Office (NKFIH)2020-1.1.6-JOVO-2021-00010 Hungary
CitationJournal: Protein Sci / Year: 2025
Title: Ligand binding Pro-miscuity of acylpeptide hydrolase, structural analysis of a detoxifying serine hydrolase.
Authors: Anna J Kiss-Szemán / Luca Takács / Imre Jákli / Zoltán Bánóczi / Naoki Hosogi / Daouda A K Traore / Veronika Harmat / András Perczel / Dóra K Menyhárd /
Abstract: Acylpeptide hydrolase (APEH) or acylaminoacyl-peptidase (AAP) is a serine hydrolase that regulates protein metabolism. It can also bind to and process unusual substrates, acting as a detoxifier. To ...Acylpeptide hydrolase (APEH) or acylaminoacyl-peptidase (AAP) is a serine hydrolase that regulates protein metabolism. It can also bind to and process unusual substrates, acting as a detoxifier. To better understand its promiscuous specificity, we determined the cryo-EM structures of mammalian APEH complexed with classical serine protease partners: a chloromethyl-ketone (CMK) inhibitor, an organophosphate (OP) pesticide (dichlorvos), and benzenesulfonyl-fluoride. Since CMK derivatives of N-acetylated peptides were suggested to induce apoptosis by inhibiting APEH, while OP complexes may serve as biomarkers of OP exposure and are linked to cognitive enhancement, these complexes carry physiological significance. We identified a unique strand-breaker Pro residue in the hydrolase domain, which relaxes the active site into a partially inactivated but more spacious conformation, transforming the classical serine protease apparatus into a versatile yet potent hydrolysis center with broad specificity, distinguishing the mammalian enzyme not only from other APEHs but also from serine α/β hydrolases sharing essentially the same fold.
History
DepositionJan 8, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)326,1118
Polymers325,2984
Non-polymers8134
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Acylamino-acid-releasing enzyme / AARE / Acyl-peptide hydrolase / APH / Acylaminoacyl-peptidase


Mass: 81324.391 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P19205, acylaminoacyl-peptidase
#2: Chemical
ChemComp-AES / 4-(2-AMINOETHYL)BENZENESULFONYL FLUORIDE / AEBSF


Mass: 203.234 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H10FNO2S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: Tetrameric structure of acylaminoacyl-peptidase (AAP) in covalent complex with AES hydrolysed from AEBSF
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Sus scrofa (pig)
Buffer solutionpH: 7.5
Buffer componentConc.: 10 mM / Name: TRIS / Formula: TRIS
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: OTHER / Nominal magnification: 165000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 12 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1
EM imaging opticsEnergyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
7PHENIXmodel fitting
11cryoSPARC4.6.0classification
12cryoSPARC3D reconstruction
13PHENIX1.20.1_4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169418 / Num. of class averages: 10 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 7px8

7px8


Accession code: 7px8 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.8 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221320
ELECTRON MICROSCOPYf_angle_d0.43729104
ELECTRON MICROSCOPYf_dihedral_angle_d3.6342980
ELECTRON MICROSCOPYf_chiral_restr0.0423284
ELECTRON MICROSCOPYf_plane_restr0.0043712

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