NMR assignments for Ca2+-free Tb24 were described previously20 (BMRB no 18011). These assignments served as a basis for obtaining nuclear Overhauser effect (NOE) distances, hydrogen-bonds, and dihedral angle restraints from the NMR data used as input for calculating the atomic-resolution structure by restrained molecular dynamics simulations as described in the Methods section. Table I summarizes the structure statistics for the 20 lowest energy conformers (Protein Databank accession no. 2lvv). The calculated structures were validated using PROCHECK, which shows that 79.1% of the residues belong to the most favorable region in the Ramachandran plot.
The final NMR-derived structures of Ca2+-free Tb24 are illustrated in Figure 2 and summarized in Table I. The 20 lowest energy conformers when superimposed have an overall main chain root mean square deviation (RMSD) of 0.7 Å (Supporting Information Fig. S1). The Tb24 main chain structure contains a total of eight α-helices and four β-strands: α1(residues 26–38), α2(residues 47–60), α3(residues 70–79), α4(residues 90–105), α5(residues 119–139), α6(residues 152–164), α7(residues 172–182), α8(residues 189–204), β1(residues 67–69), β2(residues 116–118), β3(residues 149–151), and β4(residues 186–188). Tb24 contains four EF hands: EF1 (green residues 22–55), EF2 (red, residues 61–91), EF3 (cyan residues 101–130), and EF4 (yellow, residues 140–167). The four EF-hands associate into two pairs through a two-stranded β-sheet arrangement: EF1 (green) pairs with EF2 (red), while EF3 (cyan) pairs with EF4 (yellow). The two pairs of EF-hands are connected by a long, central helix (α5) formed by merging the exiting helix of EF2 into the entering helix of EF3. This topology causes three long, central helices (α4, α5, and α8) to interact closely as a twisted, vertical bundle, causing contact between EF2 and EF4. The overall main chain structure of Tb24 is very similar to that of T. cruzi FCaBP (RMSD is 1.0 Å when comparing main chain atoms). An important structural difference between Tb24 and FCaBP can be seen in the exposed groove region [see dashed curve in Fig. 2(A)] that is lined by hydrophobic residues (V108, V175, C194, and T198) that are not conserved in FCaBP.
Figure 2. NMR-derived structure of Tb24. The average main chain structure is shown by a ribbon diagram (A) and space-filling representations (B and C). N-terminal residues (1–26) are unstructured and not shown. In panel (A), EF-hands are colored as defined in Figure 1 and the exposed hydrophobic groove is marked by a dashed curved. In panels (B,C), acidic residues (Asp and Glu), basic residues (Arg, His, Lys), and hydrophobic residues (Ile, Leu, Phe, Met, Val) are colored red, blue, and yellow, respectively. The surface in panel (B) is proposed to interact with the flagellar membrane, whereas the surface in panel (C) (hydrophobic groove) is postulated to bind to protein targets. Protein databank accession no. 2lvv.
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