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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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Structural Characterization of EhCaBP1 bound to Phenylalanine molecule.(A)Structure of trimeric EhCaBP1 showing the phenylalanine molecule bound in the hydrophobic pocket formed by the assembled domain. Three molecules interact with each other by head-tail manner forming an assembled domain at the interface. (B) Close view of assembled domain showing phenylalanine in the hydrophobic pocket surrounded by several hydrophobic residues. (C) 2Fo-Fc electron density map of phenylalanine at 2σ level. (D) The N-terminal domain of CaM-peptide complex is superposed on the assembled domain of EhCaBP1-phenylalanine complex. The hydrophobic hankering residue of the peptide in CaM-peptide complex is bound to similar location as Phe in the assembled domain of EF1 of one molecule (hot pink) and EF2 of neighboring molecule (deep teal).
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pone-0005269-g006: Structural Characterization of EhCaBP1 bound to Phenylalanine molecule.(A)Structure of trimeric EhCaBP1 showing the phenylalanine molecule bound in the hydrophobic pocket formed by the assembled domain. Three molecules interact with each other by head-tail manner forming an assembled domain at the interface. (B) Close view of assembled domain showing phenylalanine in the hydrophobic pocket surrounded by several hydrophobic residues. (C) 2Fo-Fc electron density map of phenylalanine at 2σ level. (D) The N-terminal domain of CaM-peptide complex is superposed on the assembled domain of EhCaBP1-phenylalanine complex. The hydrophobic hankering residue of the peptide in CaM-peptide complex is bound to similar location as Phe in the assembled domain of EF1 of one molecule (hot pink) and EF2 of neighboring molecule (deep teal).

Mentions: The two EF hand motifs belonging to N-terminal domain of EhCaBP1 are separated by long helix. In contrast, the corresponding EF motifs in CaM are connected by a short loop, thus bringing these two EF hand motifs into close proximity and forming a two EF-hand domain. The N-terminal domain of three molecules of EhCaBP1 participates in domain swapping to form trimers (Figure 6A) [10]. This allows the EF1-hand motif of one molecule to interact with EF2 of an adjacent molecule to form a two EF-hand domain. This assembled domain is similar to that of the two EF hand domains of CaM and TnC. This is essentially facilitated by a couple of critical residues in the linker that separate EF1 and EF2 motifs in comparison to CaM and ELC's [10]. CaM and CaM-like proteins bind to their targets by anchoring hydrophobic residue of the target. Two EF hand motifs of each domain of CaM bind to one hydrophobic residue of the target. To understand the target binding mode, EhCaBP1 was co-crystallized with Phenylalanine (Phe). Crystal structure of EhCaBP1 with Phe showed that the hydrophobic pocket formed at the interface between EF1 and EF2 in the assembled domain is bound to Phe (Figure 6B) with good electron density (Figure 6C). The Phe forms several hydrophobic interactions with Ile 8, Phe 24, Val 21 and Val25 residues of EF hand motif 1 of one molecule and Tyr 61, Phe 60, Phe 57, Ile 40 and Leu 37 residues of EF hand motif 2 of another molecule (Figure 6C).


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)

Structural Characterization of EhCaBP1 bound to Phenylalanine molecule.(A)Structure of trimeric EhCaBP1 showing the phenylalanine molecule bound in the hydrophobic pocket formed by the assembled domain. Three molecules interact with each other by head-tail manner forming an assembled domain at the interface. (B) Close view of assembled domain showing phenylalanine in the hydrophobic pocket surrounded by several hydrophobic residues. (C) 2Fo-Fc electron density map of phenylalanine at 2σ level. (D) The N-terminal domain of CaM-peptide complex is superposed on the assembled domain of EhCaBP1-phenylalanine complex. The hydrophobic hankering residue of the peptide in CaM-peptide complex is bound to similar location as Phe in the assembled domain of EF1 of one molecule (hot pink) and EF2 of neighboring molecule (deep teal).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005269-g006: Structural Characterization of EhCaBP1 bound to Phenylalanine molecule.(A)Structure of trimeric EhCaBP1 showing the phenylalanine molecule bound in the hydrophobic pocket formed by the assembled domain. Three molecules interact with each other by head-tail manner forming an assembled domain at the interface. (B) Close view of assembled domain showing phenylalanine in the hydrophobic pocket surrounded by several hydrophobic residues. (C) 2Fo-Fc electron density map of phenylalanine at 2σ level. (D) The N-terminal domain of CaM-peptide complex is superposed on the assembled domain of EhCaBP1-phenylalanine complex. The hydrophobic hankering residue of the peptide in CaM-peptide complex is bound to similar location as Phe in the assembled domain of EF1 of one molecule (hot pink) and EF2 of neighboring molecule (deep teal).
Mentions: The two EF hand motifs belonging to N-terminal domain of EhCaBP1 are separated by long helix. In contrast, the corresponding EF motifs in CaM are connected by a short loop, thus bringing these two EF hand motifs into close proximity and forming a two EF-hand domain. The N-terminal domain of three molecules of EhCaBP1 participates in domain swapping to form trimers (Figure 6A) [10]. This allows the EF1-hand motif of one molecule to interact with EF2 of an adjacent molecule to form a two EF-hand domain. This assembled domain is similar to that of the two EF hand domains of CaM and TnC. This is essentially facilitated by a couple of critical residues in the linker that separate EF1 and EF2 motifs in comparison to CaM and ELC's [10]. CaM and CaM-like proteins bind to their targets by anchoring hydrophobic residue of the target. Two EF hand motifs of each domain of CaM bind to one hydrophobic residue of the target. To understand the target binding mode, EhCaBP1 was co-crystallized with Phenylalanine (Phe). Crystal structure of EhCaBP1 with Phe showed that the hydrophobic pocket formed at the interface between EF1 and EF2 in the assembled domain is bound to Phe (Figure 6B) with good electron density (Figure 6C). The Phe forms several hydrophobic interactions with Ile 8, Phe 24, Val 21 and Val25 residues of EF hand motif 1 of one molecule and Tyr 61, Phe 60, Phe 57, Ile 40 and Leu 37 residues of EF hand motif 2 of another molecule (Figure 6C).

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