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N- and C-terminal domains of the calcium binding protein EhCaBP1 of the parasite Entamoeba histolytica display distinct functions.

Jain R, Kumar S, Gourinath S, Bhattacharya S, Bhattacharya A - PLoS ONE (2009)

Bottom Line: The results suggest that the N-terminal domain retains some of the properties, such as localization in phagocytic cups and activation of kinase.The C-terminal domain did not show any of the activities tested.The results suggest that the two domains of EhCaBP1 are functionally and structurally different from each other.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

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 Ca(2+) 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.

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Cloning and Expression of EhCaBP1 domains.(A) Schematic representation of EhCaBP1 domains. Nter protein lacks the carboxy terminus of the protein and contained only the initial 66 amino acids, while Cter protein lacks the initial 66 amino acids. (B) The induction and purification profiles for both the Nter and Cter domains are shown. Induced lysates from the bacterial cells expressing the EhCaBP1 domains are resolved on a 15% SDS-PAGE and the gel is stained with Coomassie Brilliant Blue R-250.
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pone-0005269-g001: Cloning and Expression of EhCaBP1 domains.(A) Schematic representation of EhCaBP1 domains. Nter protein lacks the carboxy terminus of the protein and contained only the initial 66 amino acids, while Cter protein lacks the initial 66 amino acids. (B) The induction and purification profiles for both the Nter and Cter domains are shown. Induced lysates from the bacterial cells expressing the EhCaBP1 domains are resolved on a 15% SDS-PAGE and the gel is stained with Coomassie Brilliant Blue R-250.

Mentions: The nucleotide sequences encoding the two domains were separately cloned in Escherichia coli expression vector pET 3(c) as described in “materials and methods”. The amino terminal domain (Nter) contained amino acids 1–66 and the carboxy terminal domain (Cter) contained amino acids 67–134 (Figure 1A). The integrity of each construct was checked by nucleotide sequencing. The domains were expressed in presence of the inducer IPTG and the expressed proteins were analysed by SDS-gel electrophoresis (Figure 1B). Purification of the expressed proteins from E. coli was carried out essentially as described before [4]. The results show that the Cter domain is expressed at a higher level compared to the Nter domain. At higher concentrations, the domains were found to be less soluble compared to the whole protein (data not shown here).


N- and C-terminal domains of the calcium binding protein EhCaBP1 of the parasite Entamoeba histolytica display distinct functions.

Jain R, Kumar S, Gourinath S, Bhattacharya S, Bhattacharya A - PLoS ONE (2009)

Cloning and Expression of EhCaBP1 domains.(A) Schematic representation of EhCaBP1 domains. Nter protein lacks the carboxy terminus of the protein and contained only the initial 66 amino acids, while Cter protein lacks the initial 66 amino acids. (B) The induction and purification profiles for both the Nter and Cter domains are shown. Induced lysates from the bacterial cells expressing the EhCaBP1 domains are resolved on a 15% SDS-PAGE and the gel is stained with Coomassie Brilliant Blue R-250.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2668073&req=5

pone-0005269-g001: Cloning and Expression of EhCaBP1 domains.(A) Schematic representation of EhCaBP1 domains. Nter protein lacks the carboxy terminus of the protein and contained only the initial 66 amino acids, while Cter protein lacks the initial 66 amino acids. (B) The induction and purification profiles for both the Nter and Cter domains are shown. Induced lysates from the bacterial cells expressing the EhCaBP1 domains are resolved on a 15% SDS-PAGE and the gel is stained with Coomassie Brilliant Blue R-250.
Mentions: The nucleotide sequences encoding the two domains were separately cloned in Escherichia coli expression vector pET 3(c) as described in “materials and methods”. The amino terminal domain (Nter) contained amino acids 1–66 and the carboxy terminal domain (Cter) contained amino acids 67–134 (Figure 1A). The integrity of each construct was checked by nucleotide sequencing. The domains were expressed in presence of the inducer IPTG and the expressed proteins were analysed by SDS-gel electrophoresis (Figure 1B). Purification of the expressed proteins from E. coli was carried out essentially as described before [4]. The results show that the Cter domain is expressed at a higher level compared to the Nter domain. At higher concentrations, the domains were found to be less soluble compared to the whole protein (data not shown here).

Bottom Line: The results suggest that the N-terminal domain retains some of the properties, such as localization in phagocytic cups and activation of kinase.The C-terminal domain did not show any of the activities tested.The results suggest that the two domains of EhCaBP1 are functionally and structurally different from each other.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

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 Ca(2+) 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.

Show MeSH
Related in: MedlinePlus