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

Abstract

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 FCaBPE151Q/E188Q and recombinant wildtype and mutant proteins as well. Both FCaBP and FCaBPE151Q/E188Q were found to associate with lipid rafts, indicating the Ca2+-independence of this association. To our initial surprise, FCaBPE151Q/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 FCaBPE151Q/E188Q, like some other Ca2+ sensors, may form dimers and that dimerization of FCaBPE151Q/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.