A novel metalloprotease family discovered through a tardigrade

Tardigrades possess numerous proteins with poorly understood functions, including candidates proposed to have been acquired through horizontal gene transfer. Here, we characterized RvY_14254 from Ramazzottius varieornatus , previously designated ATPM5, whose homologs are widely distributed across bacteria and eukaryotes but remain largely uncharacterized. We determined the crystal structure of the protein, hereafter termed Rv Peti, at 1.35 Å resolution. Rv Peti adopts a compact, C-shaped architecture with no close structural homologs in the Protein Data Bank and contains a conserved His-Asp-Asp (HDD) metal-binding motif within a deep surface pocket. Structural comparison revealed partial similarity of the active-site architecture to that of metalloproteases, including pseudoalterin. Anomalous diffraction analyses demonstrated that the HDD site can accommodate Zn 2+ and Mn 2+ . Biochemical assays showed that Rv Peti exhibits Zn 2+ dependent proteolytic activity, which was strongly reduced by a metal chelating reagent and by alanine substitutions of the metal-coordinating residues. In contrast, only weak esterase activity was detected, with the substrates tested. These results identify Rv Peti as a founding member of a previously uncharacterized family of metalloproteases and provide a structural framework for investigating the physiological and evolutionary roles of this widespread protein family.