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

J Biol Chem. 2018 Jul 13;293(28):11154-11165. doi: 10.1074/jbc.RA118.003244. Epub 2018 May 23.


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 Clostridium difficile (PPEP-1) cleaves two C. difficile cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the ppep-1 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 Paenibacillus alvei, 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.

Keywords: PPEP; Paenibacillus alvei; Pro-Pro endopeptidase; bacterial adhesion; cell wall; metalloprotease; structural biology; substrate specificity; virulence factor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Crystallography, X-Ray
  • Dipeptides / chemistry
  • Dipeptides / metabolism*
  • Endopeptidases / chemistry
  • Endopeptidases / metabolism*
  • Models, Molecular
  • Paenibacillus / enzymology*
  • Paenibacillus / growth & development
  • Protein Conformation
  • Sequence Homology
  • Substrate Specificity


  • Bacterial Proteins
  • Dipeptides
  • prolyl-proline
  • Endopeptidases

Associated data

  • PDB/5A0X
  • PDB/5A0P
  • PDB/5A0R
  • PDB/5A0S
  • PDB/2N6J
  • PDB/6FPC