# Structural analyses and substrate profiling of PPEP-3 provide new insights into the molecular basis of Pro-Pro endopeptidase specificity

## TL;DR

This study reveals how PPEP-3 cleaves proline-proline bonds by combining structural analysis and substrate profiling.

## Contribution

The study identifies unique structural features in PPEP-3 that determine its distinct substrate specificity.

## Key findings

- PPEP-3's substrate specificity differs notably at the P2 and P2′ positions compared to other PPEPs.
- Key residues Tyr161 and Phe191 shape the substrate-binding cleft of PPEP-3.
- Combining structural and substrate data provides insights into protease function.

## Abstract

Pro-Pro endopeptidases (PPEPs) are secreted bacterial enzymes that uniquely cleave peptide bonds between adjacent proline residues. Their active site accommodates six substrate residues (P3 to P3′), with interactions at these positions determining specificity. In this study, we investigated the substrate specificity of PPEP-3 from Geobacillus thermodenitrificans using synthetic peptide libraries and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We also determined the atomic structures of PPEP-3 in unbound and substrate-bound forms. By correlating substrate profiling with structural data, we identified key mechanisms influencing PPEP-3 specificity. This integrated analysis reveals stark differences in specificity for the P2 and P2′ positions compared to other PPEPs, most notably Tyr161 and Phe191, which shape the substrate-binding cleft and influence the accommodation of side chains at these positions. Combining comprehensive substrate profiling with structural analyses offers a powerful approach to uncover the molecular basis of protease function.

•Atomic structure of Pro-Pro endopeptidase 3 in unbound and substrate-bound forms•The substrate specificity of PPEP-3 differs from other PPEPs•Analysis of the PPEP-3 structure reveals key determinants of PPEP-3 specificity

Atomic structure of Pro-Pro endopeptidase 3 in unbound and substrate-bound forms

The substrate specificity of PPEP-3 differs from other PPEPs

Analysis of the PPEP-3 structure reveals key determinants of PPEP-3 specificity

Biological sciences; biochemistry; structural biology; proteomics; methodology in biological sciences