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- PDB-8pba: Cryo-EM structure of Caenorhabditis elegans DPF-3 (apo) -

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Basic information

Entry
Database: PDB / ID: 8pba
TitleCryo-EM structure of Caenorhabditis elegans DPF-3 (apo)
ComponentsDipeptidyl Peptidase Four (IV) family
KeywordsHYDROLASE / Dipeptidylpeptidase
Function / homology: / Dipeptidylpeptidase IV, N-terminal domain / Dipeptidyl peptidase IV (DPP IV) N-terminal region / Peptidase S9, prolyl oligopeptidase, catalytic domain / Prolyl oligopeptidase family / serine-type peptidase activity / Alpha/Beta hydrolase fold / proteolysis / Dipeptidyl Peptidase Four (IV) family
Function and homology information
Biological speciesCaenorhabditis elegans (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsGudipati, R.K. / Cavadini, S. / Kempf, G. / Grosshans, H.
Funding support Switzerland, Poland, 4items
OrganizationGrant numberCountry
Swiss National Science Foundation182880 Switzerland
Other private
Polish National Science CentreSONATA BIS 2021/42/E/NZ1/00336 Poland
Polish National Science CentreOPUS 2022/45/B/NZ2/02183 Poland
CitationJournal: Mol Syst Biol / Year: 2024
Title: Deep quantification of substrate turnover defines protease subsite cooperativity.
Authors: Rajani Kanth Gudipati / Dimos Gaidatzis / Jan Seebacher / Sandra Muehlhaeusser / Georg Kempf / Simone Cavadini / Daniel Hess / Charlotte Soneson / Helge Großhans /
Abstract: Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to ...Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify >40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4's active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications.
History
DepositionJun 8, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 15, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _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.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dipeptidyl Peptidase Four (IV) family
B: Dipeptidyl Peptidase Four (IV) family


Theoretical massNumber of molelcules
Total (without water)212,9262
Polymers212,9262
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Dipeptidyl Peptidase Four (IV) family


Mass: 106462.812 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: dpf-3, CELE_K02F2.1, K02F2.1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q965K3
Has protein modificationN

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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: Homodimeric complex of DPF-3 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
1RELIONparticle selection
4GctfCTF correction
5cryoSPARCCTF correction
6RELIONCTF correction
9Cootmodel fitting
10ISOLDEmodel fitting
12RosettaEMmodel refinement
13cryoSPARCinitial Euler assignment
15RELIONclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 223429 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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