We describe the largest proteolytic assembly in nature, reaching up to ~10 micron in length of thousands of copies of metalloprotease domains in bacterial flagella. Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. We report the bioinformatics-driven discovery of a new class of enzymatic flagellins that assemble to form proteolytically active flagella.
Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this new flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (we termed flagellinolysin) is the second most abundant protein in the flagella. We constructed a high confidence structural model based on the available crystal structures of clostridial collagenases. Remarkably, all of the identified flagellins conserve the critical Zn++ ion-binding histidine residues in the HEXXH catalytic motif—the glutamate being the general base, as well as additional key residues in close proximity to the active site, including the more distal third zinc ion ligating glutamate and Ala265 forming the metalloprotease hydrophobic “basement".
To identify flagellinolysin cleavage sites we employed PICS (Proteome-wide Identification of Protease Cleavage Specificity), a proteomic technique that employs proteome-derived peptide libraries as proteolytic substrate screens. After incubation with either recombinant flagellin hypervariable region (flagellinolysin), or with intact flagella, the neo amino-termini were biotinylated, affinity isolated, and identified by LC-MS/MS. Flagellinolysin and flagella cleaved nearly 1,000 different peptides. Purified flagellar filaments and recombinant flagellin exhibit metalloproteolytic activity on quenched fluorescent synthetic peptides designed from these data. By electron microscopy we localized flagellinolysin to the extracellular flagellar surface. With ~20,000 flagellin copies per ~10-μm flagella this assembles the largest proteolytic complex known revealing this family as enzymatic biopolymers with potential for numerous roles in saprophytic bacteria and in pathogens.