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

Abstract

, 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.