NMR Derived Solution Structure of an EF-Hand Calcium-Binding Protein from Entamoeba Histolytica

Abstract

We present the three-dimensional (3D) solution structure of a calcium-binding protein from Entamoeba histolytica (EhCaBP), an etiologic agent of amoebiasis affecting millions worldwide. EhCaBP is a 14.7 kDa (134 residues) monomeric protein thought to play a role in the pathogenesis of amoebiasis. The 3D structure of Ca(2+)-bound EhCaBP has been derived using multidimensional nuclear magnetic resonance (NMR) spectroscopic techniques. The study reveals the presence of two globular domains connected by a flexible linker region spanning 8 amino acid residues. Each domain consists of a pair of helix-loop-helix motifs similar to the canonical EF-hand motif of calcium-binding proteins. EhCaBP binds to four Ca(2+) with high affinity (two in each domain), and it is structurally related to calmodulin (CaM) and troponin C (TnC) despite its low sequence homology ( approximately 29%) with these proteins. NMR-derived structures of EhCaBP converge within each domain with low RMSDs and angular order-parameters for backbone torsion angles close to 1.0. However, the presence of a highly flexible central linker region results in an ill-defined orientation of the two domains relative to one other. These findings are supported by backbone (15)N relaxation rate measurements and deuterium exchange studies, which reveal low structural order parameters for residues in the central linker region. Earlier, biochemical studies showed that EhCaBP is involved in a novel signal transduction mechanism, distinct from CaM. A possible reason for such a functional diversity is revealed by a detailed 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 water exposed total hydrophobic surface area as compared to CaM and TnC. Further 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 crucial 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 acid composition is required for the function of the protein.

Functional Manipulation of a Calcium-binding Protein from Entamoeba histolytica Guided by Paramagnetic NMR

“…The presence of such a large number of CaBPs in this organism suggests that E. histolytica has an intricate and extensive Ca 2ϩ signaling system. Three of these proteins: EhCaBP1 (PDB code 1jfk) (8), EhCaBP2 (PDB code 2jnx) (9) and EhCaM (PDB code 2lc5) (10) have been structurally characterized by NMR in our laboratory and were shown to have unique individual properties. EhCaBP1 has been studied more extensively and its role in phagocytosis has been deciphered (8, 10 -12).…”

“…EhCaBP1 (14.8 kDa) is the first Ca 2ϩ -binding protein identified and structurally characterized from E. histolytica (8). The structure of EhCaBP1 consists of two domains separated by a linker and each domain consists of two EF-hand motifs (8,14).…”

“…Recent studies revealed that the two domains of EhCaBP1 are functionally and structurally different from each other (15,16) but both domains are required for structural stability and a full range of functional diversity (17). Although there is low sequence identity between EhCaBP1 and CaM, the structural topology of EhCaBP1 is similar to that of CaM (PDB code 1cll) and troponin C (TnC; PDB code 2jnf) (8). Furthermore, it is found to be functionally different from CaM as it binds to a different set of proteins compared with CaM (18).…”

“…The inter-domain linker of EhCaBP1 is more flexible compared with that of CaM and TnC due to the presence of 3 Gly residues in the linker region and thus EhCaBP1 shows different target specificity compared with CaM and TnC (8). In addition, the hydrophobic packing, inside the core of the protein, may also influence the target specificity of EhCaBP1.…”

“…In addition, the hydrophobic packing, inside the core of the protein, may also influence the target specificity of EhCaBP1. A closer examination of the NMR structure revealed that the N-terminal domain is structurally more rigid compared with its C-terminal counterpart (8,15,16 89 , and Glu 96 ) in the EF III binding loop as opposed to the presence of only three such residues in rest of the loops (I, II, and IV) or due to the structural differences between the N-and C-terminal domains. Furthermore, based on the amino acid sequence analysis of EhCaBP1, it was reported that the Tyr residue at the Ϫ4th position (Tyr 81 ) of the third Ca 2ϩ -binding loop (instead of a highly conserved Phe residue seen at this position in CaM and TnC) could be the cause of preferential Ca 2ϩ displacement from EF-hand III (20,21).…”