Structural insights into membrane targeting by the flagellar calcium-binding protein (FCaBP), a myristoylated and palmitoylated calcium sensor in Trypanosoma cruzi.
The flagellar calcium-binding protein (FCaBP) of the protozoan Trypanosoma cruzi is targeted to the flagellar membrane where it regulates flagellar function and assembly. As a first step toward understanding the Ca(2+)-induced conformational changes important for membrane-targeting, we report here the x-ray crystal structure of FCaBP in the Ca(2+)-free state determined at 2.2A resolution. The first 17 residues from the N terminus appear unstructured and solvent-exposed. Residues implicated in membrane targeting (Lys-19, Lys-22, and Lys-25) are flanked by an exposed N-terminal helix (residues 26-37), forming a patch of positive charge on the protein surface that may interact electrostatically with flagellar membrane targets. The four EF-hands in FCaBP each adopt a "closed conformation" similar to that seen in Ca(2+)-free calmodulin. The overall fold of FCaBP is closest to that of grancalcin and other members of the penta EF-hand superfamily. Unlike the dimeric penta EF-hand proteins, FCaBP lacks a fifth EF-hand and is monomeric. The unstructured N-terminal region of FCaBP suggests that its covalently attached myristoyl group at the N terminus may be solvent-exposed, in contrast to the highly sequestered myristoyl group seen in recoverin and GCAP1. NMR analysis demonstrates that the myristoyl group attached to FCaBP is indeed solvent-exposed in both the Ca(2+)-free and Ca(2+)-bound states, and myristoylation has no effect on protein structure and folding stability. We propose that exposed acyl groups at the N terminus may anchor FCaBP to the flagellar membrane and that Ca(2+)-induced conformational changes may control its binding to membrane-bound protein targets.
Center for Advanced Research in Biotechnology, University of Maryland, National Institute of Standards and Technology, Rockville, Maryland 20850, USA.
Jennifer N
JN
Wingard
Jane
J
Ladner
Murugendra
M
Vanarotti
Andrew J
AJ
Fisher
Howard
H
Robinson
Kathryn T
KT
Buchanan
David M
DM
Engman
James B
JB
Ames
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D019281:Dimerization; D005407:Flagella / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009227:Myristic Acids / Q000737:chemistry; D019308:Palmitic Acid / Q000737:chemistry; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry*; D014349:Trypanosoma cruzi / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D009227:Myristic Acids; D015800:Protozoan Proteins; C061478:flagellar calcium-binding protein, Trypanosoma cruzi; D019308:Palmitic Acid; D008239:Lysine