Discovery of a new Pro-Pro endopeptidase, PPEP-2, provides mechanistic insights into the differences in substrate specificity within the PPEP family

Abstract

Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen (PPEP-1) cleaves two cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family. Here, we report on the homolog of PPEP-1 in, which we denoted PPEP-2. We found that PPEP-2 is a secreted metalloprotease, which likewise cleaved a cell-surface protein encoded by an adjacent gene. However, the cleavage motif of PPEP-2, PLP↓PVP, is distinct from that of PPEP-1 (VNP↓PVP). As a result, an optimal substrate peptide for PPEP-2 was not cleaved by PPEP-1 and vice versa. To gain insight into the specificity mechanism of PPEP-2, we determined its crystal structure at 1.75 Å resolution and further confirmed the structure in solution using small-angle X-ray scattering (SAXS). We show that a four-amino-acid loop, which is distinct in PPEP-1 and -2 (GGST in PPEP-1 and SERV in PPEP-2), plays a crucial role in substrate specificity. A PPEP-2 variant, in which the four loop residues had been swapped for those from PPEP-1, displayed a shift in substrate specificity toward PPEP-1 substrates. Our results provide detailed insights into the PPEP-2 structure and the structural determinants of substrate specificity in this new family of PPEP proteases.

Molecular determinants of the mechanism and substrate specificity of Clostridium difficile proline-proline endopeptidase-1

“…For example, in PPEP-2 from Paenibacillus alvei it is replaced by a threonine. PPEP-2 does not favor an asparagine in P2 position but a leucine (19), and Lys-101 is replaced by an arginine. Whether this leads to different interactions is unclear due to the lack of a substrate complex structure of PPEP-2.…”

Environmentally Friendly Quinolones Design for a Two-Way Choice between Biotoxicity and Genotoxicity through Double-Activity 3D-QSAR Model Coupled with the Variation Weighting Method

“…It maintains the osmotic balance of the cells and reduces damage, acting as a reactive oxygen scavenging agent in the meantime [ 39 , 40 ]. In this study, the Protein Data Bank (PDB) database was used to obtain the structures of the aforementioned two enzymes found in plants, namely SOD (PDB ID: 1B06) from sour sandalwood leaves [ 41 ] and cytoplasmic peptidase Pro–Pro (PPEP-2, PDB ID: 6FPC) [ 42 ].…”

Post-Proline Cleaving Enzymes (PPCEs): Classification, Structure, Molecular Properties, and Applications

“…The zinc ion is bound to three histidine residues: His140, His144, and His150 ( Figure 5 ). Additionally, Pro-Pro endopeptidase 1, PPEP-1 (M34.002, 5a0p), and Pro-Pro endopeptidase 2, PPEP-2 (M34.003, 6fpc) shared a very similar structure with an α/β N-terminal domain (NTD) comprising twisted four-stranded β-sheet and three α-helices (α1-α3), and an α C-terminal domain (CTD) with four α-helices (α5-α8) [ 19 , 84 ]. The active site of Pro-Pro endopeptidase 2 is located at the α4-helix that separates NTD and CTD domains.…”