Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein.
The 24 kDa flagellar calcium binding protein (FCaBP) of the protozoan Trypanosoma cruzi is a calcium-acyl switch protein. FCaBP is modified by the addition of myristate and palmitate at its amino terminal segment and both modifications are required for calcium-modulated flagellar membrane association. FCaBP has four sequence motifs for potential calcium binding, and comparison to other calcium-acyl switch proteins, such as recoverin, suggested that only two of these sites are functional. Because it is not possible to predict with certainty the calcium binding affinity or selectivity based on motif analysis alone, we determined the quantitative calcium binding activity of FCaBP by direct ligand binding using the flow dialysis method. The results demonstrated the presence of two calcium binding sites in the full length FCaBP and in a mutant (FCaBPdelta12) lacking the amino terminal pair of sites. FCaBPdelta12 retains its ability to localize to the flagellum. A mutant FCaBP lacking the two carboxyl-terminal sites (FCaBPdelta34), did not bind calcium with high affinity and selectivity under the conditions used. The calcium binding properties of FCaBP are therefore distinct from other myristoyl switch proteins such as recoverin. The results add to a growing body of knowledge about the correlation of sequence motifs with calcium binding activity. Moreover, they demonstrate the need to determine the apparently novel mechanism by which FCaBP undergoes calcium modulated flagellar membrane association and its relation to calcium signal transduction.
Department of Biochemistry and Molecular Biology, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
R A
RA
Maldonado
S
S
Mirzoeva
L M
LM
Godsel
T J
TJ
Lukas
S
S
Goldenberg
D M
DM
Watterson
D M
DM
Engman
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005136:Eye Proteins; D017353:Gene Deletion; D005822:Genetic Vectors; D051597:Hippocalcin; D008074:Lipoproteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D051603:Recoverin; D014349:Trypanosoma cruzi / Q000235:genetics / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C061478:flagellar calcium-binding protein, Trypanosoma cruzi; D051603:Recoverin; D051597:Hippocalcin; D002118:Calcium