Pro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen 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 a... More
Pro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen 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 Zn 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 .
