title: Mechanistic and structural studies on legumain explain its zymogenicity, distinct activation pathways, and regulation.
abstract: The cysteine protease legumain plays important functions in immunity and cancer at different cellular locations, some of which appeared conflicting with its proteolytic activity and stability. Here, we report crystal structures of legumain in the zymogenic and fully activated form in complex with different substrate analogs. We show that the eponymous asparagine-specific endopeptidase activity is electrostatically generated by pH shift. Completely unexpectedly, the structure points toward a hidden carboxypeptidase activity that develops upon proteolytic activation with the release of an activation peptide. These activation routes reconcile the enigmatic pH stability of legumain, e.g., lysosomal, nuclear, and extracellular activities with relevance in immunology and cancer. Substrate access and turnover is controlled by selective protonation of the S1 pocket (KM) and the catalytic nucleophile (kcat), respectively. The multibranched and context-dependent activation process of legumain illustrates how proteases can act not only as signal transducers but as decision makers.
authors.affiliation: Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria.
authors.forename: Elfriede
authors.initials: E
authors.lastname: Dall
authors.forename: Hans
authors.initials: H
authors.lastname: Brandstetter
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation / Q000235:genetics; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D000099240:Asparaginyl Endopeptidase
chemical_list: D003546:Cysteine Endopeptidases; D004792:Enzyme Precursors; D000099240:Asparaginyl Endopeptidase
keywords: allostery; context-dependent activities; death domain; electrostatic stability switch; kcat-substrate specificity