Structural and Biochemical Characterization of an Atypical α-Carbonic Anhydrase from the Tardigrade Ramazzottius varieornatus

Abstract

The tardigrade Ramazzottius varieornatus exhibits extraordinary resilience to extreme environmental stresses, yet the functional diversity of its proteome remains largely unexplored. In this study, the structural and biochemical characterization of RvCA5, an atypical alpha-carbonic anhydrase (CA) identified in R. varieornatus, is presented. Expression analysis in E. coli revealed the spontaneous formation of a truncated RvCA5 species, which was confirmed to be unrelated to signal peptide cleavage. RvCA5 exhibited distinct structural features, including extended intrinsically disordered regions (IDRs) at both termini. Unlike canonical alpha-CAs, RvCA5 exhibited negligible CO2 hydration activity, which was partially enhanced by the removal of the N-terminal IDR, suggesting that this region acts as a dynamic entropic barrier hindering substrate diffusion. RvCA5 possesses multiple surface-exposed reactive cysteine residues, resembling the redox-sensing human CA 3. Notably, consistent with a predicted nuclear localization signal, in silico modeling predicted that RvCA5 can bind DNA via a positively charged patch near the C-terminal IDR. The DNA-binding capability of RvCA5 was experimentally demonstrated by electrophoretic mobility shift assays. Collectively, these findings suggest that RvCA5 potentially functions as a redox-responsive transcriptional regulator.