Redox-dependent activity and thioredoxin interaction of cyclophilin TgCyp21 from Toxoplasma gondii.

Cyclophilins (Cyps) are ubiquitous cytosolic proteins with peptidyl-prolyl cis-trans isomerase (PPIase) activity and the ability to bind the immunosuppressant cyclosporin A (CsA). The genome of Toxoplasma gondii, the parasite responsible for toxoplasmosis, encodes multiple putative Cyps, whose specific functions remain largely unexplored. Here, we characterize TgCyp21, a predicted Cyp from T. gondii. TgCyp21 displays PPIase activity and is inhibited by CsA in vitro. Importantly, its activity decreases markedly under oxidizing conditions but is partially restored by reducing agents, including dithiothreitol (DTT) and the parasite endogenous thioredoxin (TgTrx). TgCyp21 contains four cysteines, with Cys87 and Cys141 predicted to be spatially close based on structural modeling. Substitution of both residues significantly reduced PPIase activity, with Cys87 emerging as the main contributor to this loss. Structural modeling further indicates that Cys87 and Cys141 are suitably oriented to interact with the conserved active-site cysteines of TgTrx. This interaction is supported experimentally by mixed disulfide trapping, which identifies a stable disulfide-linked intermediate between TgCyp21 and TgTrx, consistent with a thiol-disulfide exchange mechanism. Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy further confirm the formation of the complex. Taken together, our data indicate that TgCyp21 behaves in vitro as a redox-responsive Cyp and a substrate for Trx, suggesting a potential involvement in Trx-mediated redox processes in T. gondii.