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

Abstract

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. 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. Flagellar calcium-binding protein (FCaBP) 3The abbreviations used are:FCaBPflagellar calcium-binding proteinHSQCheteronuclear single quantum coherenceHMQCheteronuclear multiple quantum coherenceNOEnuclear Overhauser effectNOESYNOE spectroscopyr.m.s.d.root-mean-squared deviationMES4-morpholineethanesulfonic acidPEFpenta EF-hand. is a 24-kDa highly immunogenic protein found in the flagellum of the protozoan parasite Trypanosoma cruzi (1Engman D.M. Krause K.H. Blumin J.H. Kim K.S. Kirchhoff L.V. Donelson J.E. J. Biol. Chem. 1989; 264: 18627-18631Abstract Full Text PDF PubMed Google Scholar). FCaBP contains four EF-hand calcium binding motifs (2Moncrief N.D. Kretsinger R.H. Goodman M. J. Mol. Evol. 1990; 30: 522-562Crossref PubMed Scopus (337) Google Scholar, 3Ikura M. Trends Biochem. Sci. 1996; 21: 14-17Abstract Full Text PDF PubMed Scopus (603) Google Scholar) (see Fig. 1), the third and fourth (EF-3 and EF-4) of which bind calcium (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The protein is modified at the N terminus by covalent attachment of myristate at Gly-2 and palmitate at Cys-4, both of which are required for association with the inner leaflet of the flagellar membrane (5Godsel L.M. Engman D.M. EMBO J. 1999; 18: 2057-2065Crossref PubMed Scopus (124) Google Scholar). Calcium is required for stable flagellar localization as well, as FCaBP can be washed out of detergent-permeabilized trypanosomes if calcium chelators are included in the wash solutions. The N-terminal acylation and calcium-dependent membrane localization of FCaBP suggested that the protein may possess a functional calcium-acyl switch, similar to the Ca2+-myristoyl switch observed previously for recoverin (6Zozulya S. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11569-11573Crossref PubMed Scopus (294) Google Scholar, 7Dizhoor A.M. Chen C.K. Olshevskaya E. Sinelnikova V.V. Phillipov P. Hurley J.B. Science. 1993; 259: 829-832Crossref PubMed Scopus (170) Google Scholar) and other members of the neuronal calcium sensor family (8Burgoyne R.D. Biochim. Biophys. Acta. 2004; 1742: 59-68Crossref PubMed Scopus (49) Google Scholar). Acyl switch proteins undergo calcium-dependent membrane association by virtue of calcium-regulated extrusion or sequestration of a myristate moiety that mediates membrane binding (9Ames J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (430) Google Scholar). However, an FCaBP mutant unable to bind calcium still maintains its flagellar localization, suggesting that FCaBP may not cycle the membrane on and off like some calcium acyl switch proteins (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). flagellar calcium-binding protein heteronuclear single quantum coherence heteronuclear multiple quantum coherence nuclear Overhauser effect NOE spectroscopy root-mean-squared deviation 4-morpholineethanesulfonic acid penta EF-hand. The best studied calcium myristoyl switch protein is recoverin, a calcium-binding protein in retinal rod cells that inhibits rhodopsin kinase only at high Ca2+ levels (10Kawamura S. Nature. 1993; 362: 855-857Crossref PubMed Scopus (323) Google Scholar, 11Klenchin V.A. Calvert P.D. Bownds M.D. J. Biol. Chem. 1995; 270: 16147-16152Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and regulates the recovery phase of phototransduction (12Erickson M.A. Lagnado L. Zozulya S. Neubert T.A. Stryer L. Baylor D.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6474-6479Crossref PubMed Scopus (62) Google Scholar, 13Gray-Keller M.P. Polans A.S. Palczewski K. Detwiler P.B. Neuron. 1993; 10: 523-531Abstract Full Text PDF PubMed Scopus (138) Google Scholar). In the resting dark state, recoverin binds two calcium ions and associates with retinal rod outer segment membranes through its exposed myristoyl group (6Zozulya S. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11569-11573Crossref PubMed Scopus (294) Google Scholar, 7Dizhoor A.M. Chen C.K. Olshevskaya E. Sinelnikova V.V. Phillipov P. Hurley J.B. Science. 1993; 259: 829-832Crossref PubMed Scopus (170) Google Scholar). Photoexcitation of the rod cell results in a lowering of cytosolic calcium (14Koch K.W. Stryer L. Nature. 1988; 334: 64-66Crossref PubMed Scopus (473) Google Scholar), causing recoverin to lose its bound calcium and adopt a conformation in which the myristoyl group becomes sequestered within a hydrophobic cleft in the protein (9Ames J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (430) Google Scholar). Ca2+-free recoverin then dissociates from rhodopsin kinase at the membrane, allowing rhodopsin kinase to phosphorylate light-excited rhodopsin and promote receptor inactivation. The calcium-myristoyl switch mechanism allows calcium regulation of two proteins (rhodopsin kinase and rhodopsin) that do not themselves bind calcium. One major difference between FCaBP and recoverin is the presence of palmitate in FCaBP, which may or may not participate in a potential switch mechanism. A variety of FCaBP-like proteins are found in other trypanosomes: Trypanosoma rangeli, Trypanosoma lewisi, and Trypanosoma brucei (Fig. 1). A flagellar calmodulin has also been characterized in T. brucei (15Ridgley E. Webster P. Patton C. Ruben L. Mol. Biochem. Parasitol. 2000; 109: 195-201Crossref PubMed Scopus (34) Google Scholar). These Ca2+-binding proteins all localize to the flagellum, a unique organelle that has many functions, including motility, chemotaxis, and cell signaling. In addition to the traditional “9 + 2” microtubule structure of the axoneme, there is a structure as the rod that the The axoneme, and are by the flagellar has been by analysis that the flagellar membrane contains a of the membrane L. Google Scholar), and the flagellum of T. brucei is highly in Flagellar membrane and function is also by the of FCaBP to the membrane where it is to interact with a variety of membrane-bound protein (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). We report here the x-ray crystal structure of Ca2+-free FCaBP as a first step toward understanding the Ca2+-induced conformational changes that control membrane is only the structure of a switch protein in the Ca2+-free The structure of Ca2+-free recoverin first its covalently attached myristoyl group to be highly sequestered and the protein T. Ames J.B. Stryer L. Ikura M. Nature. 1995; PubMed Scopus Google Scholar). the crystal structure of Ca2+-bound a sequestered myristoyl group R. M. Palczewski K. Full Text Full Text PDF PubMed Scopus Google Scholar). the structure of Ca2+-free FCaBP in suggests a solvent-exposed N its covalently attached acyl groups to interact with membrane targets. We propose that the exposed acyl groups and N-terminal residues (Lys-19, and may promote the binding of FCaBP to the flagellar membrane and that Ca2+-induced protein conformational changes may control its binding to membrane-bound protein targets. and FCaBP FCaBP with a in a of the as previously (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) in with or to 1989; PubMed Scopus Google Scholar, Biophys. 1990; PubMed Scopus Google Scholar, J.B. Tanaka T. Stryer L. Ikura M. PubMed Scopus Google Scholar). FCaBP for the crystal as in and and with and to a at FCaBP with a to a of and FCaBP and to in in a with a FCaBP by FCaBP and in cells on with acid and FCaBP from the of cell on a the then to an in A and with a at of the of to and to in as by by the J. Kim S.H. J. Scopus Google Scholar). by of and with of and a and The protein crystal in a with the The with of protein and of at for the structure crystal a The The crystal to with a of and by the crystal through The with Biol. 1999; PubMed Scopus Google Scholar). are in for the protein at on the and with 1997; PubMed Scopus Google Scholar). the in group group only and the as the in group The to The Biol. PubMed Scopus Google Scholar, Scopus Google Scholar) used to and the of the A for the that there two in the for group or for The used in Biol. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar) to an and the in P. K. Biol. 2004; PubMed Scopus Google Scholar) and the which of and by and by Biol. 1997; Scopus Google Scholar), of the residues and The by in and in The to the and in both group and The and the the no to for the the for the group are in The residues The of residues with and as The has four the seen and a conformation for The region including residues has the is In a of the residues the and the as by of of of unique of in a of of in a NMR for NMR analysis of FCaBP with and a covalently myristoyl FCaBP protein for NMR in of a and or NMR at on a with a and with and a with an a and a with The (see Fig. on a of FCaBP The of and The (see Fig. and on a of FCaBP protein attached to myristate J.B. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) as as FCaBP bound to myristate not as previously C. PubMed Scopus Google N-terminal myristoyl group of FCaBP to be solvent-exposed. NMR of acid in and the myristoyl group of Ca2+-free and Ca2+-bound The myristoyl group with and the protein The in the attached to acyl NOE as previously R. T. PubMed Scopus Google Scholar). out with a of by the FCaBP in the with of FCaBP in the is seen to be in the as in by NMR and The structure from on crystal the presence of and crystal appear for in the structure suggesting that has no effect on the overall In each crystal the first 17 residues from the N terminus appear acid of the protein from that all 17 N-terminal residues in both crystal not by x-ray crystal structure of Ca2+-free FCaBP at with a of and of (Fig. The has been for the first 17 residues from the N for the N-terminal residues may be NMR and heteronuclear nuclear Overhauser effect analysis on FCaBP also that the first 17 N-terminal residues are indeed unstructured not and the N-terminal myristoyl group is solvent-exposed (see The overall structure for residues of FCaBP determined by x-ray with and to a NMR structure The protein structure in the crystal has a fold with overall of (Fig. The first in the crystal at that the of an conformation (residues a that a with an (residues N-terminal helix is a of four (residues (residues (residues (residues FCaBP contains a of and four (residues (residues (residues (residues (residues (residues (residues (residues (residues (residues and (residues (Fig. 1). The four EF-hands of FCaBP two through a with with The two of EF-hands are by a helix by the helix of the helix of and to interact as a As a and with of EF-hands is similar to that seen for the first four EF-hands in the proteins, grancalcin J. K. M. J. Mol. Biol. 2000; PubMed Scopus Google Scholar) and of P. M. Mol. Biol. 1997; Scopus Google Scholar). However, proteins, FCaBP lacks a fifth EF-hand and is not EF-hands and Ca2+ EF-hands in FCaBP each of a structure similar to the conformation of EF-hands seen in Ca2+-free of calmodulin M.A. M. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Tanaka T. Ikura M. Biol. 1995; PubMed Scopus Google Scholar), S. Biol. 1995; PubMed Scopus Google Scholar), and grancalcin J. K. M. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). The for the EF-hands in FCaBP are and The binding of Ca2+ to each EF-hand in calmodulin a in that of an to the of many hydrophobic residues that interact with protein Ikura M. Full Text Full Text PDF PubMed Scopus Google Scholar, E. J. Biochem. 270: PubMed Scopus Google Scholar). Ca2+ binding to and in FCaBP (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) may a similar Ca2+-induced of hydrophobic residues and the observed Ca2+-induced binding of FCaBP to protein and other protein (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). membrane binding by FCaBP in by its Ca2+-induced binding to protein on the flagellar membrane The Ca2+ binding of in FCaBP in crystal and an conformation in crystal which may is not to bind Ca2+ (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The of the may be in to the presence of at in the binding which lacks an required for Ca2+ at The of Ca2+ binding at may also Ca2+ is not to bind to as the of and to a causing both EF-hands to be in the Ca2+-free In at in the is not to a with which may the structure and Ca2+ binding at and both adopt a conformation for binding with functional Ca2+ binding at (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In the Ca2+ binding of from residues and are in a a bound (Fig. that the binding of Ca2+ with the M. Trends Biochem. Sci. 1996; 21: 14-17Abstract Full Text PDF PubMed Scopus (603) Google Scholar). from residues and are also with a similar that a bound In the of the in the Ca2+ binding of and both with at the forming a conformation of Ca2+-bound calmodulin and C. the Ca2+ binding in and both adopt a conformation that promote and functional Ca2+ of of FCaBP multiple residues in FCaBP on of the protein surface (Fig. with an exposed hydrophobic patch on the (Fig. is the of N-terminal and residues (Lys-19, and and a of positive charge on the protein surface in in Fig. of N-terminal residues (Lys-19, Lys-22, and Lys-25) been implicated previously in membrane We propose that the exposed patch of N-terminal and residues may promote with groups on the surface of the flagellar A similar membrane has also been for the myristoyl switch proteins, recoverin K. M. Ikura M. Ames J.B. S. PubMed Scopus Google Scholar) and the protein S. A. Trends Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). The membrane by the N-terminal myristoyl group and can the membrane binding by S. A. Trends Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar, M.D. Full Text PDF PubMed Scopus Google Scholar). The protein surface on the (Fig. contains an exposed patch of hydrophobic residues the terminus and in in Fig. The exposed hydrophobic residues are on the of and Ca2+-induced conformational changes in the and of hydrophobic We propose that the exposed hydrophobic patch in Fig. may a binding for the with membrane-bound protein targets. NMR of of from the first 17 residues of FCaBP suggested that the N-terminal region and myristoyl group may be the of the myristoyl we NMR of both and FCaBP, that both are (Fig. the presence of the myristoyl group not appear to the overall structure of Ca2+-free FCaBP, in contrast to the changes in Ca2+-free recoverin that from N-terminal myristoylation T. Ames J.B. Stryer L. Ikura M. Nature. 1995; PubMed Scopus Google Scholar, J.B. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The in the of FCaBP and suggests that the acyl may be exposed to not with the NMR also on FCaBP a myristoyl group to the and of the N-terminal myristoyl and NMR on of recoverin that a myristoyl group used to the the N-terminal myristoyl group J.B. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, T. Ames J.B. M. Stryer L. Ikura M. J. 1998; PubMed Scopus Google Scholar). These that the covalently attached acyl in recoverin is sequestered in a hydrophobic in the Ca2+-free protein and that binding of Ca2+ to conformational changes that the group NMR on of FCaBP that a myristoyl group (Fig. the that are covalently attached to only the and of the acyl appear in and are to from the The of the group in Ca2+-free FCaBP (Fig. similar to the of acid in (Fig. and the of are of the myristoyl group for Ca2+-free FCaBP (Fig. from of for which determined previously J.B. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The in the of the myristoyl group in Ca2+-free FCaBP to that of acid in that in Ca2+-free FCaBP the acyl is solvent-exposed, in contrast to Ca2+-free recoverin where the myristoyl is the of the protein (9Ames J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (430) Google Scholar). the myristoyl group of FCaBP is solvent-exposed, and J.B. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) on FCaBP protein a These of residues in the protein that within of the group of the myristoyl NOE between the myristate group and the protein not be not The of in suggests that the group of the acyl is from in the for the that the group of FCaBP is solvent-exposed and not interact with the The addition of Ca2+ to no in the NMR of the myristoyl group (Fig. In NOE between the myristate and protein not be not in and of Ca2+-bound suggesting that the group of the acyl not interact with the Ca2+-bound the myristoyl group of Ca2+-bound FCaBP solvent-exposed and not undergo a in in contrast to has been observed previously in recoverin J.B. Tanaka T. Ikura M. Stryer L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). with EF-hand N-terminal myristoylation and four EF-hands in FCaBP that it be similar to recoverin (9Ames J.B. Ishima R. Tanaka T. Gordon J.I. Stryer L. Ikura M. Nature. 1997; 389: 198-202Crossref PubMed Scopus (430) Google Scholar, Zozulya S. Stryer L. 1993; Full Text PDF PubMed Scopus Google Scholar) and Ca2+-myristoyl switch proteins J. V.V. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Biol. 1999; PubMed Scopus Google Scholar). However, the overall of FCaBP and recoverin are and the overall fold of Ca2+-free FCaBP is similar to that of J. K. M. J. Mol. Biol. 2000; PubMed Scopus Google Scholar) and proteins that to the family M. S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). FCaBP to the proteins The deviation the of the four EF-hands of FCaBP with of the first four EF-hands of grancalcin (Fig. The is the from and the structure of between the two The helix of and first helix of in FCaBP are a helix that is flanked in an by and forming a A similar and overall is also seen in grancalcin and to be a of all The of the between FCaBP and proteins suggests that be grancalcin and proteins Ca2+-induced membrane binding Biochem. J. 1992; PubMed Scopus Google Scholar, C. A.P. E. 1995; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar) and interact with proteins as K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (49) Google Scholar), A.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and A. D.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) implicated in FCaBP also binds to membranes and has been to interact with proteins (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). are to of the FCaBP proteins be to or other protein that and important difference between FCaBP and proteins is that FCaBP is in and not a fifth EF-hand like the dimeric The fifth EF-hand of FCaBP may been to for the of hydrophobic residues in (Fig. that be if the fifth EF-hand difference is the presence of the N-terminal region in that implicated in membrane targeting M. K. Biochem. J. 1997; Google Scholar). FCaBP has residues (Lys-19, Lys-22, Lys-25) in the N-terminal region that we be important for membrane binding in addition to N-terminal myristoylation and and We propose that the four EF-hands of are to the first four EF-hands of proteins and that may a of the family that lacks a fifth EF-hand and N-terminal myristoylation and motifs be to for other of proteins that are to FCaBP and as calcium crystal structure of FCaBP (Fig. has for its Ca2+-induced mechanism (Fig. The solvent-exposed myristoylation (5Godsel L.M. Engman D.M. EMBO J. 1999; 18: 2057-2065Crossref PubMed Scopus (124) Google at the N terminus that the covalently attached acyl groups as membrane that the In exposed residues in the N-terminal region (Fig. are suggested to binding by electrostatically with groups on the membrane A similar of and myristoylation seen previously for membrane binding by the switch of and kinase protein S. A. Trends Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar). NMR and that the acyl group is exposed of Ca2+ suggesting that both the Ca2+-free and Ca2+-bound of FCaBP may be to the flagellar membrane (Fig. then is membrane binding by FCaBP by We propose that the Ca2+ localization of FCaBP at the flagellar membrane be by binding of FCaBP to proteins that bind to the Ca2+-free or Ca2+-bound of FCaBP (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The binding of Ca2+ to calmodulin and other EF-hand proteins to the of hydrophobic residues that binding to protein Ikura M. Full Text Full Text PDF PubMed Scopus Google Scholar, E. J. Biochem. 270: PubMed Scopus Google Scholar). A similar of hydrophobic residues in (Fig. promote association of Ca2+-bound FCaBP with a membrane-bound protein and localize FCaBP at the flagellar membrane at high Ca2+ levels (Fig. the Ca2+-free state of FCaBP bind to a cytosolic protein that localize FCaBP in the at Ca2+ levels (Fig. The binding of a cytosolic protein to Ca2+-free FCaBP to the N-terminal acyl groups a protein exposed hydrophobic residues in (Fig. like that seen for Ca2+-free recoverin T. Ames J.B. Stryer L. Ikura M. Nature. 1995; PubMed Scopus Google Scholar) or R. M. Palczewski K. Full Text Full Text PDF PubMed Scopus Google Scholar). of the myristoyl group cytosolic FCaBP promote its membrane only at Ca2+ of the group attached to FCaBP a may its membrane The of palmitate may also be by In both the Ca2+-free and Ca2+-bound FCaBP exposed myristoyl groups (Fig. that we propose anchor the protein to the flagellar FCaBP Ca2+-induced extrusion of the myristoyl group to control membrane localization like is seen for recoverin (6Zozulya S. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11569-11573Crossref PubMed Scopus (294) Google Scholar) and Ca2+-myristoyl switch proteins R.D. Biochem. J. PubMed Scopus Google Scholar). we propose that Ca2+-induced protein conformational changes in FCaBP (4Buchanan K.T. Ames J.B. Asfaw S.H. Wingard J.N. Olson C.L. Campana P.T. Araujo A.P. Engman D.M. J. Biol. Chem. 2005; 280: 40104-40111Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) may its with proteins that control membrane localization as in Fig. on Ca2+-bound FCaBP bound to proteins are to and the mechanism. We for with NMR for NMR for the E. FCaBP and and and for for NMR and