Abstract
Tripeptidyl peptidase II (TPP II) is the largest known eukaryotic protease (6 MDa). It is believed to act downstream of the 26S proteasome, cleaving tripeptides from the N termini of longer peptides, and it is implicated in numerous cellular processes. Here we report the structure of Drosophila TPP II determined by a hybrid approach. We solved the structure of the dimer by X-ray crystallography and docked it into the three-dimensional map of the holocomplex, which we obtained by single-particle cryo-electron microscopy. The resulting structure reveals the compartmentalization of the active sites inside a system of chambers and suggests the existence of a molecular ruler determining the size of the cleavage products. Furthermore, the structure suggests a model for activation of TPP II involving the relocation of a flexible loop and a repositioning of the active-site serine, coupling it to holocomplex assembly and active-site sequestration.