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- PDB-8rfb: Cryo-EM structure of the R243C mutant of human Prolyl Endopeptida... -

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Basic information

Entry
Database: PDB / ID: 8rfb
TitleCryo-EM structure of the R243C mutant of human Prolyl Endopeptidase-Like (PREPL) protein involved in Congenital myasthenic syndrome-22 (CMS22)
ComponentsProlyl endopeptidase-like
KeywordsHYDROLASE / thio-esterase
Function / homology
Function and homology information


Golgi to plasma membrane protein transport / retrograde transport, endosome to Golgi / regulation of synaptic vesicle exocytosis / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / trans-Golgi network / peptidase activity / cytoskeleton / serine-type endopeptidase activity / Golgi apparatus / proteolysis ...Golgi to plasma membrane protein transport / retrograde transport, endosome to Golgi / regulation of synaptic vesicle exocytosis / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / trans-Golgi network / peptidase activity / cytoskeleton / serine-type endopeptidase activity / Golgi apparatus / proteolysis / nucleus / cytosol
Similarity search - Function
: / Peptidase S9A, prolyl oligopeptidase / Peptidase S9A, N-terminal domain / Prolyl oligopeptidase, N-terminal beta-propeller domain / Peptidase S9, prolyl oligopeptidase, catalytic domain / Prolyl oligopeptidase family / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Prolyl endopeptidase-like
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.01 Å
AuthorsTheodoropoulou, A. / Cavani, E. / Antanasijevic, A. / Marcaida, M.J. / Dal Peraro, M.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: JCI Insight / Year: 2024
Title: Missense variants in CMS22 patients reveal that PREPL has both enzymatic and nonenzymatic functions.
Authors: Yenthe Monnens / Anastasia Theodoropoulou / Karen Rosier / Kritika Bhalla / Alexia Mahy / Roeland Vanhoutte / Sandra Meulemans / Edoardo Cavani / Aleksandar Antanasijevic / Irma Lemmens / ...Authors: Yenthe Monnens / Anastasia Theodoropoulou / Karen Rosier / Kritika Bhalla / Alexia Mahy / Roeland Vanhoutte / Sandra Meulemans / Edoardo Cavani / Aleksandar Antanasijevic / Irma Lemmens / Jennifer A Lee / Catherine J Spellicy / Richard J Schroer / Ricardo A Maselli / Chamindra G Laverty / Patrizia Agostinis / David J Pagliarini / Steven Verhelst / Maria J Marcaida / Anne Rochtus / Matteo Dal Peraro / John Wm Creemers /
Abstract: Congenital myasthenic syndrome-22 (CMS22, OMIM 616224) is a rare genetic disorder caused by deleterious genetic variation in the prolyl endopeptidase-like (PREPL) gene. Previous reports have ...Congenital myasthenic syndrome-22 (CMS22, OMIM 616224) is a rare genetic disorder caused by deleterious genetic variation in the prolyl endopeptidase-like (PREPL) gene. Previous reports have described patients with deletions and nonsense variants in PREPL, but nothing is known about the effect of missense variants in the pathology of CMS22. In this study, we have functionally characterized missense variants in PREPL from 3 patients with CMS22, all with hallmark phenotypes. Biochemical evaluation revealed that these missense variants do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria and disease mechanism. Structural analysis showed that the variants affect regions most likely involved in intraprotein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the 3 variants. The importance of nonhydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines, which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the transgolgi network. In conclusion, these studies showed that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense variants that do not necessarily result in a loss of hydrolytic activity of PREPL.
History
DepositionDec 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.title / _em_admin.last_update
Revision 1.2Oct 9, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Prolyl endopeptidase-like


Theoretical massNumber of molelcules
Total (without water)73,4771
Polymers73,4771
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area28710 Å2
MethodPISA

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Components

#1: Protein Prolyl endopeptidase-like / Prolylendopeptidase-like


Mass: 73477.398 Da / Num. of mol.: 1 / Mutation: R243C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PREPL, KIAA0436 / Production host: Escherichia coli (E. coli)
References: UniProt: Q4J6C6, Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
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: cryoEM structure of the R243C mutant of prolyl endopeptidase-like (PREPL) protein
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHepes1
31 mMTris-(2-Carboxyethyl)phosphine, HydrochlorideTCEP1
SpecimenConc.: 13 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 4 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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: cryoSPARC / Version: 3.2.0 / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 137436 / Symmetry type: POINT

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