NMR derived solution structure of an EF-hand calcium-binding protein from Entamoeba histolytica

We present the three-dimensional (3D) solution structure of a calcium-bindi ng protein from Entamoeba histolytica (EhCaBP), an etiologic agent of amoeb iasis affecting millions worldwide. EhCaBP is a 14.7 kDa ( 134 residues) mo nomeric protein thought to play a role in the pathogenesis of amoebiasis. T he 3D structure of Ca2+-bound EhCaBP has been derived using, multidimension al nuclear magnetic resonance (NMR) spectroscopic techniques. The study rev eals the presence of two globular domains connected by a flexible linker re gion spanning 8 amino acid residues. Each domain consists of a pair of heli x-loop-helix motifs similar to the canonical EF-hand motif of calcium-bindi ng proteins. EhCaBP binds to four Ca2+ with high affinity (two in each doma in), and it is structurally related to calmodulin (Cam) and troponin C (TnC ) despite its low sequence homology (similar to 29%) with these proteins. N MR-derived structures of EhCaBP converge within each domain with low RMSDs and angular order-parameters for backbone torsion angles close to 1.0. Howe ver, the presence of a highly flexible central linker region results in an ill-defined orientation of the two domains relative to one other. These fin dings are supported by backbone N-15 relaxation rate measurements and deute rium exchange studies, which reveal low structural order parameters for res idues in the central linker region. Earlier, biochemical studies showed tha t EhCaBP is involved in a novel signal transduction mechanism, distinct Fro m CaM. A possible reason for such a functional diversity is revealed by a d etailed comparison of the 3D structure of EhCaBP with that of CaM and TnC. The studies indicate a more open C-terminal domain for EhCaBP with larger w ater exposed total hydrophobic surface area as compared to CaM and TnC. Fur ther dissimilarities between the structures include the presence of two Gly residues (G63 and G67) in the central linker region of EhCaBP, which seem to impart it a greater flexibility compared to CaM and TnC and also play cr ucial role in its biological function. Thus, unlike in CaM and TnC, wherein the length and/or composition of the central linker have been found to be crucial for their function, in EhCaBP, both flexibility as well as amino ac id composition is required for the function of the protein.