PXD061585

PXD061585 is an original dataset announced via ProteomeXchange.

Title	Biochemical and structural characterization of PPEP-3 from Geobacillus thermodenitrificans
Description	The members of the group of Pro-Pro endopeptidases (PPEPs) are secreted bacterial endoproteases that display a unique preference for hydrolyzing their substrates between two proline residues. The active site cleft of PPEPs accommodates the six substrate residues P3 to P3’, and the interactions between these and the protease sites S3 to S3’ determine PPEP specificity beyond the scissile Pro-Pro peptide bond. In this study, we have characterized the substrate specificity of PPEP-3 from the thermophilic bacterium Geobacillus thermodenitrificans in detail using synthetic combinatorial peptide libraries in combination with LC-MS/MS analyses. There is a stark difference for the P2 and P2’ positions compared to PPEP-1: instead of Asn and Val at P2, either Ser or basic residues are preferred, while Pro is almost exclusively tolerated at P2’. In the crystal structure of substrate-bound PPEP-3, most notably Tyr161 and Phe191, which differ from the corresponding residues in PPEP-1 (histidine and leucine, respectively), greatly influence the P2 and P2’ specificity. By correlating the substrate specificity profile to the structure, we explore the various mechanisms that determine PPEP-3 specificity and highlight differences with other PPEPs.
HostingRepository	PRIDE
AnnounceDate	2025-11-11
AnnouncementXML	Submission_2025-11-11_03:53:24.082.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Bart Claushuis
SpeciesList	scientific name: Geobacillus thermodenitrificans; NCBI TaxID: NEWT:33940;
ModificationList	biotinylated residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2025-03-07 08:35:54	ID requested
⏵ 1	2025-11-11 03:53:24	announced

Dataset with its publication pending

submitter keyword: Geobacillus thermodenitrificans, Macromolecular X-ray crystallography, zinc metallopeptidase, Mass spectrometry, substrate specificity,Pro-Pro Endopeptidase-3

Peter A. van Veelen
contact affiliation	Center for Proteomics and Metabolomics, Leiden University Medical Center, The Netherlands
contact email	p.a.van_veelen@lumc.nl
lab head
Bart Claushuis
contact affiliation	Leiden University Medical Center
contact email	b.claushuis@lumc.nl
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/11/PXD061585

PRIDE project URI

• PRIDE
  1. PXD061585
     1. Label: PRIDE project
     2. Name: Biochemical and structural characterization of PPEP-3 from Geobacillus thermodenitrificans