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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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Ability of EhCaBP1 domains to activate endogenous kinase(s).(A) E. histolytica cell-free lysate (25 µg) is used as the source of kinase and histone type III (15 µg) as the substrate. EhCaBP1, Nter or Cter (2 nM) is added to the reaction mixture at varying concentration and the assay is performed using [γ-32P] ATP as the phosphate donor. The concentration of Ca2+ used is 10 µM. The reaction products are separated on a 12% SDS-PAGE, air dried and autoradiography is done. The first lane is histone alone. (B) The amount of radioactivity incorporated into histone as determined by densitometry. The reaction mixtures are precipitated and the incorporated counts are measured using liquid scintillation counter. The graph represents the intensity measurement of three independent experiments ±s.d.
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pone-0005269-g003: Ability of EhCaBP1 domains to activate endogenous kinase(s).(A) E. histolytica cell-free lysate (25 µg) is used as the source of kinase and histone type III (15 µg) as the substrate. EhCaBP1, Nter or Cter (2 nM) is added to the reaction mixture at varying concentration and the assay is performed using [γ-32P] ATP as the phosphate donor. The concentration of Ca2+ used is 10 µM. The reaction products are separated on a 12% SDS-PAGE, air dried and autoradiography is done. The first lane is histone alone. (B) The amount of radioactivity incorporated into histone as determined by densitometry. The reaction mixtures are precipitated and the incorporated counts are measured using liquid scintillation counter. The graph represents the intensity measurement of three independent experiments ±s.d.

Mentions: EhCaBP1 is known to activate endogenous kinase(s) in a Ca2+ dependent manner [5]. The ability of the domains to activate these kinase(s) was tested as described before, using histone phosphorylation visualized by autoradiography [9] (Figure 3A). While Nter could activate the endogenous kinase more efficiently than the complete EhCaBP1, Cter showed a marked reduction in activity of about 50–60% of the control (Figure 3B).


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)

Ability of EhCaBP1 domains to activate endogenous kinase(s).(A) E. histolytica cell-free lysate (25 µg) is used as the source of kinase and histone type III (15 µg) as the substrate. EhCaBP1, Nter or Cter (2 nM) is added to the reaction mixture at varying concentration and the assay is performed using [γ-32P] ATP as the phosphate donor. The concentration of Ca2+ used is 10 µM. The reaction products are separated on a 12% SDS-PAGE, air dried and autoradiography is done. The first lane is histone alone. (B) The amount of radioactivity incorporated into histone as determined by densitometry. The reaction mixtures are precipitated and the incorporated counts are measured using liquid scintillation counter. The graph represents the intensity measurement of three independent experiments ±s.d.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005269-g003: Ability of EhCaBP1 domains to activate endogenous kinase(s).(A) E. histolytica cell-free lysate (25 µg) is used as the source of kinase and histone type III (15 µg) as the substrate. EhCaBP1, Nter or Cter (2 nM) is added to the reaction mixture at varying concentration and the assay is performed using [γ-32P] ATP as the phosphate donor. The concentration of Ca2+ used is 10 µM. The reaction products are separated on a 12% SDS-PAGE, air dried and autoradiography is done. The first lane is histone alone. (B) The amount of radioactivity incorporated into histone as determined by densitometry. The reaction mixtures are precipitated and the incorporated counts are measured using liquid scintillation counter. The graph represents the intensity measurement of three independent experiments ±s.d.
Mentions: EhCaBP1 is known to activate endogenous kinase(s) in a Ca2+ dependent manner [5]. The ability of the domains to activate these kinase(s) was tested as described before, using histone phosphorylation visualized by autoradiography [9] (Figure 3A). While Nter could activate the endogenous kinase more efficiently than the complete EhCaBP1, Cter showed a marked reduction in activity of about 50–60% of the control (Figure 3B).

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