A novel metalloprotease family discovered through a tardigrade

Abstract

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 RvPeti, at 1.35 Å resolution. RvPeti 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 Zn² and Mn² . Biochemical assays showed that RvPeti exhibits Zn² -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 RvPeti 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. statement Tardigrades, organisms capable of surviving extreme environment stress, possess numerous poorly characterized proteins. We determined the crystal structure of one such protein and found that it adopts a novel fold containing a metal-binding site. Biochemical analyses confirmed zinc-dependent proteolytic activity that was lost upon mutation of the metal-binding residues. These findings define a previously unrecognized metalloprotease family conserved across bacteria and eukaryotes, providing a structural framework for future function studies.