Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein

The 24 kDa flagellar calcium binding protein (FCaBP) of the protozoan Trypa nosoma cruzi is a calcium-acyl switch protein. FCaBP is modified by the add ition of myristate and palmitate at its amino terminal segment and both mod ifications are required for calcium-modulated flagellar membrane associatio n. FCaBP has four sequence motifs for potential calcium binding, and compar ison to other calcium-acyl switch proteins, such as recoverin, suggested th at only two of these sites are functional. Because it is not possible to pr edict with certainty the calcium binding affinity or selectivity based on m otif analysis alone, we determined the quantitative calcium binding activit y of FCaBP by direct ligand binding using the flow dialysis method. The res ults demonstrated the presence of two calcium binding sites in the full len gth FCaBP and in a mutant (FCaBP Delta 12) lacking the amino terminal pair of sites. FCaBP Delta 12 retains its ability to localize to the flagellum. A mutant FCaBP lacking the two carboxyl-terminal sites (FCaBP Delta 34), di d not bind calcium with high affinity and selectivity under the conditions used. The calcium binding properties of FCaBP are therefore distinct from o ther myristoyl switch proteins such as recoverin. The results add to a grow ing body of knowledge about the correlation of sequence motifs with calcium binding activity. Moreover, they demonstrate the need to determine the app arently novel mechanism by which FCaBP undergoes calcium modulated flagella r membrane association and its relation to calcium signal transduction. (C) 1999 Elsevier Science B.V. All rights reserved.