Distinct Calcium Binding and Structural Properties of Two Centrin Isoforms from Toxoplasma gondii

Centrins are calcium (Ca²⁺)-binding proteins that have been implicated in several regulatory functions. In the protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, three isoforms of centrin have been identified. While increasing information is now available that links the function of centrins with defined parasite biological processes, knowledge is still limited on the metal-binding and structural properties of these proteins. Herein, using biophysical and structural approaches, we explored the Ca²⁺ binding abilities and the subsequent effects of Ca²⁺ on the structure of a conserved (TgCEN1) and a more divergent (TgCEN2) centrin isoform from T. gondii. Our data showed that TgCEN1 and TgCEN2 possess diverse molecular features, suggesting that they play nonredundant roles in parasite physiology. TgCEN1 binds two Ca²⁺ ions with high/medium affinity, while TgCEN2 binds one Ca²⁺ with low affinity. TgCEN1 undergoes significant Ca²⁺-dependent conformational changes that expose hydrophobic patches, supporting a role as a Ca²⁺ sensor in toxoplasma. In contrast, Ca²⁺ binding has a subtle influence on conformational features of TgCEN2 without resulting in hydrophobic exposure, suggesting a different Ca²⁺ relay mode for this isoform. Furthermore, TgCEN1 displays a Ca²⁺-dependent ability to self-assemble, while TgCEN2 did not. We discuss our findings in the context of Ca²⁺ signaling in toxoplasma.