¹H, ¹⁵N, and ¹³C Chemical Shift Assignments of the Calflagin Tb24 Flagellar Calcium Binding Protein of Trypanosoma brucei

Abstract

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. We report complete NMR chemical shift assignments of the flagellar calcium binding protein calflagin Tb24 of T. brucei. (BMRB no. 18011).

Biological Context

Flagellar calcium-binding proteins (FCaBPs) are immunogenic proteins found in the flagellum of protozoan parasites: Trypanosoma cruzi (Engman et al., 1989), Trypanosoma brucei (Haghighat and Ruben, 1992), and Trypanosoma rangeli (Porcel et al., 1996) (see Fig. 1). FCaBPs contain four EF-hand calcium-binding motifs (Ikura, 1996; Moncrief, 1990) (Fig. 1), the third and fourth (EF-3 and EF-4) of which bind calcium (Maldonado et al., 1999). The protein is modified at the N terminus by covalent attachment of myristate at Gly2 and palmitate at Cys4, both of which along with conserved lysine residues near the N-terminus (Maric et al., 2011) are required for association with the inner leaflet of the flagellar membrane (Godsel and Engman, 1999). Calcium is required for stable flagellar localization as well, since T. cruzi 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 T. cruzi FCaBP suggested that the protein may possess a functional calcium-acyl switch, similar to the Ca²⁺-myristoyl switch observed previously for recoverin (Dizhoor et al., 1993; Zozulya and Stryer, 1992) and other members of the neuronal calcium sensor (NCS) family (Burgoyne, 2004).

Figure 1.

Alignment of the primary sequence of T. brucei calflagin Tb24 with those of T. rangeli FCaBP and T. cruzi FCaBP. Secondary structural elements indicated schematically were derived from analysis of NMR data (³J_hnhα, chemical shift index (Wishart et al., 1992) and sequential NOE patterns). The four EF-hands (EF1, EF2, EF3 and EF4) are highlighted green, salmon, cyan, and yellow, respectively. Residues in the 12-residue Ca²⁺-binding loops are underlined and chelating residues are highlighted bold. Invariant basic residues implicated in membrane binding are colored blue.

Three FCaBP homologs in T. brucei have been characterized and named the calflagins: Tb24, Tb17 and Tb44 (Wu et al., 1994). The calflagins are similar in sequence to T. cruzi FCaBP (Fig. 1) and exhibit Ca²⁺-induced localization to the flagellar membrane during cell motility, chemotaxis, and cell signaling. T. brucei calflagin Tb24 possesses different membrane targeting properties compared to that of T. cruzi FCaBP. Tb24 requires both myristoylation and palmitoylation for binding to flagellar membranes (Emmer et al., 2010; Pinto et al., 2003), whereas the unpalmitoylated Tb24 is trafficked to the pellicular (cell body) membrane (Emmer et al., 2009). This is in stark contrast to T. cruzi FCaBP, whose unpalmitoylated form is mislocalized in the cytoplasm (Godsel and Engman, 1999). The different membrane targeting properties of modified forms of these similar proteins is perhaps due to some protein structural difference between the two, or due to different lipid composition of various trypanosome membrane domains (Tyler et al., 2009). T. brucei Tb24 and T. cruzi FCaBP may also differ by interacting with distinct membrane-bound protein targets (Buchanan et al., 2005; Wingard et al., 2008).

Atomic resolution structures of T. brucei FCaBPs in both the Ca²⁺-free and Ca²⁺-bound states are needed to elucidate their Ca²⁺-induced conformational changes that control membrane-targeting, and to understand their structural differences with T. cruzi FCaBP. We report here detailed NMR resonance assignments for Ca²⁺-free calflagin Tb24 (henceforth referred to simply as Tb24) as a first step toward this end.

Methods and Experiments

Expression and Purification of Tb24.

Recombinant and uniformly ¹⁵N- or ¹⁵N/¹³C-labeled Tb24 was expressed in E. coli strain, BL21(DE3) grown on M9 medium supplemented with ¹⁵N-NH₄Cl and/or ¹³C₆-glucose. Recombinant protein expression was induced by exogenously adding 0.5 mM isopropyl β-D-l-thiogalactopyranoside (IPTG) to cells grown overnight at 25 °C. Typically, a 1-L culture yields about 20 mg of purified protein. Detailed procedures for purifying Tb24 are described elsewhere (Emmer et al., 2010).

NMR spectroscopy.

Samples of recombinant Ca²⁺-free Tb24 (0.7 mM) were prepared in 90%/10% H₂O/D₂O or 100% D₂O with 10 mM sodium phosphate (pH 7.0), 4 mM DTT-d₁₁ and 0.3 mM EDTA-d₁₂. NMR experiments were conducted using Bruker Advance 600 MHz spectrometer equipped with a triple resonance cryogenic probe. All experiments were performed at 298 K. Backbone and side-chain chemical shift assignments were obtained using ¹⁵N-HSQC, HNCO, HNCACB, CBCACONH, HBHACONH and ¹⁵N-HSQC-TOCSY (mixing time of 60 ms) spectra (Ikura et al., 1990). Methyl group side-chain resonances were assigned using ¹³C-CT-HSQC and ¹³C-HCCH-TOCSY. For aromatic side-chain chemical shift assignments, HBCBCGCDHD, HBCBCGCDCEHE, ¹³C-CT-HSQC-TOCSY spectra (Yamazaki et al., 1993) along with ¹³C-HSQC-NOESY, recorded with a mixing time of 120 ms, were used. NMR data were processed using NMRPipe (Delaglio et al., 1995) software package and analyzed using SPARKY.

Assignments and Data Deposition

Figure 2 presents HSQC spectra of Ca²⁺-free Tb24 at pH 7.0 to illustrate representative backbone resonance assignments. NMR assignments were based on 3D heteronuclear NMR experiments performed on ¹³C/¹⁵N-labeled Tb24 (residues 2–218). The protein sample in this study consists of 217 native residues with an unmyristoylated N-terminus (Gly 2) and lacking palmitoylation at Cys 3. All non-proline residues exhibited strong backbone amide resonances with uniform intensities, indicative of a well-defined three-dimensional protein structure. More than 95% of the backbone resonances (¹HN, ¹⁵N, ¹³Cα, ¹³Cβ, and ¹³CO) and ~82% of aliphatic side chain resonances were assigned. The chemical shift assignments (¹H, ¹⁵N, ¹³C) of Ca²⁺-free Tb24 have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under accession number 18011.

Figure 2.

Two-dimensional ¹⁵N-HSQC NMR spectra of Ca²⁺-free Tb24 recorded at 600 MHz proton frequency (A) and expanded view of spectrally crowded central region (B). Side chain amide resonances of Asn and Gln are connected with dotted lines. Representative resonance assignments are indicated and the complete list of assignments can be found at the BMRB repository (accession no. 18011).

The chemical shift index of each amino acid residue reveals a protein secondary structure in Tb24 similar to that observed previously for T. cruzi FCaBP (Wingard et al., 2008) (Fig. 1). Tb24 contains 8 α-helices and two antiparallel β-sheets (αl: 26–38; α2: 47–60; β1: 67–69; α3: 70–79; α4: 90–105; β2: 116–118; α5: 119–139; β3: 149–151; α6: 152–164; α7: 172–182; β4: 186–188; α8: 189–204). The NMR assignments reported here for Ca²⁺-free Tb24 are compared to those reported previously for T. cruzi FCaBP (Wingard et al., 2008) (Fig. 3). Similar chemical shifts are seen for conserved residues in the N-terminal region that might interact with membrane targets (highlighted blue in Fig. 1), suggesting that Tb24 may use dual acyl groups for membrane anchoring similar to that described previously for T. cruzi FCaBP (Godsel and Engman, 1999). The largest chemical shift differences are observed for residues in the second EF-hand (highlighted red in Fig. 3), suggesting that these residues may play a structural role in explaining the different membrane-targeting properties of Tb24 versus T. cruzi FCaBP (Emmer et al., 2010; Emmer et al., 2009; Godsel and Engman, 1999).

Figure 3.

Backbone amide ¹⁵N chemical shift difference (between Tb24 and T. cruzi FCaBP) vs. residue number. EF-hand residues are highlighted using the same color scheme in Fig. 1.