Molecular recognition of myosin L by the Toxoplasma gondii calmodulin.

Abstract

Calmodulin is a conserved EF-hand Ca2+ sensor that transduces intracellular Ca2+ signals through interactions with target proteins. Recent cryo-electron microscopy studies revealed the Toxoplasma gondii calmodulin (TgCaM) as a light chain associated with the neck of the unconventional myosin MyoL within the preconoidal P2 ring , a key component of the conoid involved in parasite motility and invasion. Here, we used NMR spectroscopy, isothermal titration calorimetry, and complementary biophysical approaches to characterize TgCaM and its interaction with a synthetic peptide spanning the predicted TgCaM-binding site in the MyoL neck. TgCaM displays the hallmark properties of canonical CaMs, undergoing a Ca2+-dependent conformational transition that exposes hydrophobic target-recognition surfaces. ITC and residue-specific NMR analyses revealed that the C-terminal EF-hand lobe binds Ca2+ with higher affinity than the N-terminal lobe, consistent with the conserved functional asymmetry of CaMs. Moreover, TgCaM binds the MyoL neck peptide with submicromolar affinity, Ca2+ dependence and 1:1 stoichiometry. Peptide binding increased the apparent Ca2+ affinity of TgCaM, revealing thermodynamic coupling between Ca2+ binding and target recognition. Together, these findings provide new insights into the biochemical and biophysical properties of the TgCaM-MyoL peptide interaction in the context of the conoid-associated complex.