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Abstract
Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, and calcium signaling is thought to be involved in amoebic pathogenesis. EhCaBP1, a Ca2+ binding protein of E. histolytica, is essential for parasite growth. High resolution crystal structure of EhCaBP1 suggested an unusual arrangement of the EF-hand domains in the N-terminal part of the structure, while C-terminal part of the protein was not traced. The structure revealed a trimer with amino terminal domains of the three molecules interacting in a head-to-tail manner forming an assembled domain at the interface with EF1 and EF2 motifs of different molecules coming close to each other. In order to understand the specific roles of the two domains of EhCaBP1, the molecule was divided into two halves, and each half was separately expressed. The domains were characterized with respect to their structure, as well as specific functional features, such as ability to activate kinase and bind actin. The domains were also expressed in E. histolytica cells along with green fluorescent protein. The results suggest that the N-terminal domain retains some of the properties, such as localization in phagocytic cups and activation of kinase. Crystal structure of EhCaBP1 with Phenylalanine revealed that the assembled domains, which are similar to Calmodulin N-terminal domain, bind to Phenylalanine revealing the binding mode to the target proteins. The C-terminal domain did not show any of the activities tested. However, over-expression in amebic cells led to a dominant negative phenotype. The results suggest that the two domains of EhCaBP1 are functionally and structurally different from each other. Both the domains are required for structural stability and full range of functional diversity.
Highlights
Calcium (Ca2+) is a ubiquitous intracellular signal responsible for controlling numerous cellular processes in wide spectrum of organisms
The results show that the carboxy terminal domain (Cter) domain is expressed at a higher level compared to the Nter domain
Ca2+ signal is perceived by a set of proteins known as calcium binding proteins
Summary
Calcium (Ca2+) is a ubiquitous intracellular signal responsible for controlling numerous cellular processes in wide spectrum of organisms. E. histolytica genome encodes a large repertoire of CaBPs as revealed by a motif-based search for EF-hand containing proteins suggesting an extensive Ca2+-based signaling network in this organism [3]. Many of these proteins are expressed in proliferating trophozoites suggesting that these are likely to be functional proteins [3, Padhan unpublished observations]. Our laboratory previously identified a 14.7 kDa calcium binding protein, EhCaBP1 [4], from E. histolytica. This protein shares 29% sequence identity with the ubiquitous CaBP, Calmodulin (CaM). The mechanism by which EhCaBP1 is recruited to the phagocytic cups is not yet clear, its ability to bind both F- and Gactin directly has been demonstrated [8]
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