The effects of free Cys residues on the structure, activity, and tetrameric stability of mammalian uricase

Mammalian uricases contain four conserved cysteine (Cys) residues, but little is known about their structures and functions. In this study, we first confirmed that all four Cys residues are free and not involved in disulfide bond formation, using canine uricase as a model protein. Cys residues had a greater effect on stability than on activity based on single Cys-to-Ser (serine) substitutions. Circular dichroism (CD) and homology modeling indicated that C188S reduces beta-sheet contents and inter- and intra-subunit hydrophobic interaction, potentially impairing the core tetrameric beta-barrel structure of the tunneling-fold protein, and ultimately decreased the tetrameric stability. Additionally, the inactivation of C188S during the stability tests may be a complex process involving depolymerization followed by irregular aggregation. Double mutations or thiol blockage of Cys188 and Cys195 significantly disrupted the formation and stability of tetrameric uricase, which may be mediated by the free thiols in Cys residues. The present results demonstrated that the free Cys residues are essential for tetrameric formation and stability in mammalian uricase. This implies that free cysteine residues, although not involved in disulfide bonding, may play important structural roles in certain proteins, underscoring the significance of the hydrophobic characteristics of the free thiols in Cys residues.

Automated summary

This study confirms the essential role of free cysteine residues in tetrameric formation and stability of mammalian uricase. Mutations or blockage of Cys188 and Cys195 significantly disrupt the formation and stability of the tetrameric structure, highlighting the importance of free thiols in Cys residues.