Abstract
The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd approximately 9 microM) and EF-4 the low affinity site (Kd approximately 120 microM). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.
Highlights
Membrane association, and calcium-dependent localization of flagellar calcium-binding protein (FCaBP), we concluded that the protein is a calcium acyl switch protein
Scatchard analysis of the curve of binding revealed two binding sites, with association constants (Ka) 9.0 Ϯ 0.9 and 122 Ϯ 1.9 M and no evidence of cooperativity. This curve shows that the complete saturation of binding sites is reached at 350 M calcium. These results are similar to calcium binding properties previously determined for another isoform of FCaBP, rC29, which binds with affinities of 3.3 Ϯ 0.5 and 190 Ϯ 0.2 M [14]
Our previous model of FCaBP posited that it is a calcium acyl switch protein that localizes to the flagellar membrane upon binding calcium and dissociates from the membrane when in the calcium-unbound state [2]
Summary
It has been shown by freeze-fracture analysis that the flagellar membrane contains a higher concentration of sterols than does the pellicular (cell body) membrane [12], and current work in our laboratory demonstrates that the flagellum of T. brucei is highly enriched in lipid rafts.3 It is especially interesting, to study the behavior of acylated calcium sensor proteins as they interact with this unique membrane. We wanted to investigate the role of calcium binding in the acyl switch mechanism by introducing point mutations in EF-3 and EF-4 to disrupt calcium binding and generated these FCaBP mutant proteins and T. cruzi epimastigotes expressing these proteins (i) to delineate the high and low affinity binding sites in FCaBP, (ii) to assess calcium-dependent conformational changes in these proteins, and (iii) to directly determine the role of calcium in the flagellar localization of FCaBP
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