Cryo-EM structures of a neofunctionalized tardigrade peroxiredoxin specialized for nucleic acid binding

Some terrestrial tardigrades can endure severe oxidative stress in part through their gene-expanded repertoire of antioxidant proteins. However, among these antioxidant proteins, Rv PrxL, a peroxiredoxin (Prx)-like protein from Ramazzottius varieornatus strain YOKOZUNA-1, is unusual in that the catalytic cysteine is replaced by glutamate, apparently incapacitating canonical peroxidase function. In this study, we investigated the structure and function of this atypical Prx. Biochemical assays demonstrated that Rv PrxL completely lacks canonical peroxidase and chaperone activities. Cryo-EM analysis revealed a unique 20-mer structure of Rv PrxL. We also observed that the tardigrade-specific N-terminal region of Rv PrxL helps retain the high-order oligomeric assembly even in the presence of highly concentrated hydrogen peroxide. The N-terminal region also promotes nuclear localization of Rv PrxL and mediates binding to nucleic acids including nuclear RNAs. Furthermore, combining a standard cryo-EM method and a new approach in which cell L ysates were Ap plied D irectly o n cryo-EM G rids (LApDoG), we visualized two distinct nucleic acid–binding modes of Rv PrxL, termed “on-ring” and “in-ring”, which likely reflect distinct physiological roles or modes of action. Collectively, these findings show that Rv PrxL has been neofunctionalized to interact with nucleic acids in the nucleus, highlighting unexpected functional diversification of antioxidant proteins in tardigrades.