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J. Biol. Chem., Vol. 283, Issue 34, 23388-23396, August 22, 2008

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Structural Insights into Membrane Targeting by the Flagellar Calcium-binding Protein (FCaBP), a Myristoylated and Palmitoylated Calcium Sensor in Trypanosoma cruzi^*

Jennifer N. Wingard¹, Jane Ladner, Murugendra Vanarotti, Andrew J. Fisher, Howard Robinson^¶, Kathryn T. Buchanan^||, David M. Engman^||, and James B. Ames²

From the Center for Advanced Research in Biotechnology, University of Maryland, National Institute of Standards and Technology, Rockville, Maryland 20850, Department of Chemistry, University of California, Davis, California 95616, ^¶Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000, and ^||Departments of Pathology and Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

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 Ca²⁺-induced conformational changes important for membrane-targeting, we report here the x-ray crystal structure of FCaBP in the Ca²⁺-free state determined at 2.2Å 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 Ca²⁺-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 Ca²⁺-free and Ca²⁺-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 Ca²⁺-induced conformational changes may control its binding to membrane-bound protein targets.

Received for publication, April 25, 2008 , and in revised form, June 12, 2008.

The atomic coordinates and structure factors (code 3CS1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported, in whole or in part, by National Institutes of Health Grants EY012347 and NS045909 (to J. B. A.), RR11973 (to the University of California Davis NMR Facility), and AI46781 (to D. M. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Current address: Dept. of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908.

² To whom correspondence should be addressed: Dept. of Chemistry, One Shields Ave., University of California, Davis, CA 95616. Tel.: 530-752-6358; Fax: 530-752-8995; E-mail: ames{at}chem.ucdavis.edu.

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