Calcium-dependent membrane association of a flagellar calcium sensor does not require calcium binding

Flagellar calcium-binding protein (FCaBP) is a dually acylated Ca²⁺ sensor in the Trypanosoma cruzi flagellar membrane that undergoes a massive conformational change upon Ca²⁺ binding. It is similar to neuronal Ca²⁺ sensors, like recoverin, which regulate their binding partners through a calcium acyl switch mechanism. FCaBP is washed out of permeabilized cells with buffers containing EDTA, indicating Ca²⁺-dependent flagellar membrane association. We hypothesized that, like recoverin, FCaBP projects its acyl groups in the presence of Ca²⁺, permitting flagellar membrane and binding partner association and that it sequesters the acyl groups in low Ca²⁺, disassociating from the membrane and releasing its binding partner to perform a presumed enzymatic function. The X-ray crystal structure of FCaBP suggests that the acyl groups are always exposed, so we set out to test our hypothesis directly. We generated T. cruzi transfectants expressing FCaBP or Ca²⁺-binding mutant FCaBP^E151Q/E188Q and recombinant wildtype and mutant proteins as well. Both FCaBP and FCaBP^E151Q/E188Q were found to associate with lipid rafts, indicating the Ca²⁺-independence of this association. To our initial surprise, FCaBP^E151Q/E188Q, like wildtype FCaBP, exhibited Ca²⁺-dependent flagellar membrane association, even though this protein does not bind Ca²⁺ itself [16]. One possible explanation for this is that FCaBP^E151Q/E188Q, like some other Ca²⁺ sensors, may form dimers and that dimerization of FCaBP^E151Q/E188Q with endogenous wildtype FCaBP might explain its Ca²⁺-dependent localization. Indeed both proteins are able to form dimers in the presence and absence of Ca²⁺. These results suggest that FCaBP possesses two distinct Ca²⁺-dependent interactions - one involving a Ca²⁺-induced change in conformation and another perhaps involving binding partner association.