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

J Biol Chem. 2019 Jul 26;294(30):11525-11535. doi: 10.1074/jbc.RA119.009029. Epub 2019 Jun 10.


Pro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen Clostridium difficile that cleaves two endogenous adhesion proteins. PPEP-1 is therefore important for bacterial motility and hence for efficient gut colonization during infection. PPEP-1 exhibits a unique specificity for Pro-Pro peptide bonds within the consensus sequence VNP↓PVP. In this study, we combined information from crystal and NMR structures with mutagenesis and enzyme kinetics to investigate the mechanism and substrate specificity of PPEP-1. Our analyses revealed that the substrate-binding cleft of PPEP-1 is shaped complementarily to the major conformation of the substrate in solution. We found that it possesses features that accept a tertiary amide and help discriminate P1' residues by their amide hydrogen bond-donating potential. We also noted that residues Lys-101, Trp-103, and Glu-184 are crucial for proteolytic activity. Upon substrate binding, these residues position a flexible loop over the substrate-binding cleft and modulate the second coordination sphere of the catalytic zinc ion. On the basis of these findings, we propose an induced-fit model in which prestructured substrates are recognized followed by substrate positioning within the active-site cleft and a concomitant increase in the Lewis acidity of the catalytic Zn2+ ion. In conclusion, our findings provide detailed structural and mechanistic insights into the substrate recognition and specificity of PPEP-1 from the common gut pathogen C. difficile.

Keywords: Clostridium difficile; crystal structure; enzyme kinetics; infectious disease; metalloprotease; nuclear magnetic resonance (NMR); protease; substrate specificity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Clostridioides difficile / enzymology*
  • Endopeptidases / chemistry
  • Endopeptidases / metabolism*
  • Hydrogen Bonding
  • Kinetics
  • Proline / chemistry*
  • Protein Conformation
  • Proteolysis
  • Substrate Specificity


  • Bacterial Proteins
  • Proline
  • Endopeptidases

Associated data

  • PDB/5A0P
  • PDB/4N4E
  • PDB/1PWV
  • PDB/5A0X
  • PDB/2QEJ