A Flagellum-specific Calcium Sensor

Abstract

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (K d ϳ9 M) and EF-4 the low affinity site (K d ϳ120 M). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation. Flagellar calcium-binding protein (FCaBP)2 is a 24-kDa highly immunogenic protein found in the flagellum of the protozoan parasite Trypanosoma cruzi (1). FCaBP contains four EF-hand calcium-binding domains, the third and fourth (EF-3 and EF-4) of which bind calcium (2). The protein is modified at the N terminus by the addition of myristate and palmitate, both of which are required for association with the inner leaflet of the flagellar membrane (3). Calcium is required for stable flagellar localization as well, since FCaBP can be washed out of detergent-permeabilized trypanosomes if calcium chelators are included in the wash solutions. Because of the size, acylation, calcium binding, membrane association, and calcium-dependent localization of FCaBP, we concluded that the protein is a calcium acyl switch protein. These proteins undergo calcium-dependent membrane association by virtue of calcium-regulated extrusion or sequestration of a myristate moiety that mediates membrane binding (4).The best studied calcium acyl switch protein is recoverin (Rv), a myristoylated calcium-binding protein of the mammalian retina (5) that inhibits rhodopsin kinase (RK) (6) and regulates recovery of the photoreceptor cell after photoexcitation (7). Rv regulates RK via the calcium myristoyl switch mechanism (4). In the resting state of the cell, Rv binds two calcium ions a...

The flagellum of Trypanosoma brucei: New tricks from an old dog

“…A single flagellum membrane-specific protein, the ESAG4 adenylate cyclase, has been identified in T. brucei (Paindavoine et al, 1992), and a calcium-binding flagellar membrane protein has been characterized in T. cruzi (Engman et al, 1989;Buchanan et al, 2005). In the flagellar matrix, several components of the intraflagellar transport pathway (IFT20, IFT52, IFT172) have recently been shown to exhibit both a soluble flagellar pool and a detergent-insoluble basal body-associated pool (Absalon et al, 2007a), but no other soluble flagellar proteins have been identified.…”

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

“…6) and of the trypanosome flagellar membrane proteome (10) have identified flagellar membrane proteins likely to be involved in signal transduction, including potential kinases, adenylate cyclases, and Ca 2ϩ channels. Indeed, the Trypanosoma cruzi Flagellar Ca 2ϩ Binding Protein (TcFCaBP) is localized specifically to the parasite flagellar membrane and has been suggested to have a role in Ca 2ϩ -dependent signal transduction (11)(12)(13). As demonstrated some years ago, the adenylate cyclase ESAG-4 is also targeted to the flagellar membrane in Trypanosoma brucei (14).…”

NMR structure of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei

“…17 T. brucei Tb24 and T. cruzi FCaBP may also differ by interacting with distinct membranebound protein targets. 18,19 Atomic resolution structures of T. brucei FCaBPs are needed to elucidate their Ca 2þ -induced conformational changes that control membrane-targeting. We report here the NMR structure for Ca 2þ -free calflagin Tb24 (henceforth referred to simply as Tb24).…”