A non-catalytic interaction surface mediates recognition of spliceosomal factors by the Toxoplasma gondii cyclophilin TgCyp23.

The spliceosome is emerging as a key regulatory hub in Toxoplasma gondii, yet the molecular basis of spliceosomal protein interactions remains largely unexplored. TgCyp23, a predicted nuclear cyclophilin (Cyp) from T. gondii, shares sequence similarity and catalytic properties with the human spliceosomal cyclophilin H (hCypH) that interacts with the splicing factors PRP4 and PRP18. Here, we investigated whether TgCyp23 engages in analogous interactions with the T. gondii orthologs TgPRP4 and TgPRP18 using peptides corresponding to their predicted Cyp-binding regions. High-resolution crystal structures of TgCyp23 in complex with a TgPRP4-derived peptide, including a ternary complex with the Cyp inhibitor cyclosporin A, reveal that spliceosomal partner recognition occurs outside the catalytic site, which remains fully accessible. NMR analyses extend this binding mode to TgPRP18, demonstrating that peptides derived from both spliceosomal factors engage the same surface in solution. Isothermal titration calorimetry shows that TgCyp23 binds both peptides with low- to mid-micromolar affinity, while circular dichroism and molecular dynamics support a folding-upon-binding mechanism. Notably, binding of either peptide does not affect the peptidyl-prolyl isomerase activity of TgCyp23 or its sensitivity to cyclosporin A, indicating that partner recognition and catalytic function are mechanistically separable. Together, these findings identify a non-catalytic interaction surface in TgCyp23 that mediates recognition of spliceosomal factors. The similar interaction mode observed for TgCyp23 and hCypH suggests that spliceosomal partner recognition may be conserved and supports a role for TgCyp23 as a spliceosome-associated Cyp in T. gondii, providing a structural framework for understanding Cyp interactions within the parasite spliceosome.