Crystal structure of calcium binding protein‐1 from Entamoeba histolytica: A novel arrangement of EF hand motifs

Abstract

Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Several EF-hand containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though these proteins have very high sequence similarity, they bind to different target proteins in a Ca2+ dependent manner, leading to different functional pathways (Yadava et al., Mol Biochem Parasito 1997;84:69-82; Chakrabarty et al., J Biol Chem 2004;279:12898-12908) The crystal structure of the Entamoeba histolytica calcium binding protein-1 (EhCaBP1) has been determined at 2.4 A resolution. The crystals were grown using MPD as precipitant and they belong to P6(3) space group with unit cell parameters of a = 95.25 A, b = 95.25 A, c = 64.99 A. Only two out of the four expected EF hand motifs could be modeled into the electron density map and the final model refined to R factor of 25.6% and Free_R of 28%. Unlike CaM, the first two EF hand motifs in EhCaBP1 are connected by a long helix and form a dumbbell shaped structure. Owing to domain swapping oligomerization three EhCaBP1 molecules interact in a head to tail manner to form a triangular trimer. This arrangement allows the EF-hand motif of one molecule to interact with that of an adjacent molecule to form a two EF-hand domain similar to that seen in the N-terminal domain of the NMR structure of CaBP1, calmodulin and troponin C. The oligomeric state of EhCaBP1 results in reduced flexibility between domains and may be responsible for the more limited set of targets recognized by EhCaBP1.

Flexibility of EF-hand motifs: structural and thermodynamic studies of Calcium Binding Protein-1 from Entamoeba histolytica with Pb2+, Ba2+, and Sr2+

“…EhCaBP1 was over-expressed and purified as previously described [19] and concentrated to 20 to 25 mg/ml. Since Pb 2+ can easily replace Ca 2+ from the calcium-binding loops of CaM, [10,11,18] Pb(NO3) 2 at an initial concentration of 1 M was directly added to the purified EhCaBP1 to bring the final concentration of Pb 2+ to 5 mM.…”

“…Hanging-drop vapor-diffusion crystallization trials were carried out under conditions similar to that used for crystallization of the native protein [19]. Crystals suitable for diffraction were obtained by mixing 5 μl of EhCaBP1-Pb complex and 2 μl of reservoir solution containing 60–65% MPD in 50 mM sodium acetate pH 3.6.…”

“…The data sets were indexed, processed and scaled using the program Automar [25]. The crystals belong to the space group P6 3 (Table 3) with two molecules per asymmetric unit, similar to that of the native structure [19] (Figure 1). …”

“…EhCaBP1 has four EF-hand motifs, each of them able to bind one calcium ion similar to that of calmodulin. The crystal structure of EhCaBP1 has been reported previously at 2.4 Å resolution by our group, where only the N-terminal half was traced and the C-terminal domain was found to be missing due to the presence of the flexible linker region between these two domains [19]. The overall structure is trimeric in which EF-hand 1 of one molecule interacts with EF-hand 2 of the other molecule in a domain swapped manner to form an assembled domain similar to that of the calmodulin N-terminal domain (Figure 1).…”

N- and C-Terminal Domains of the Calcium Binding Protein EhCaBP1 of the Parasite Entamoeba histolytica Display Distinct Functions

“…Crystal structure of EhCaBP1 showed an unusual arrangement of the domains of EhCaBP1 [10]. The region connecting EF hands I and II was found to be less flexible with extended conformation.…”