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Activation of the cardiac Na(+)-Ca(2+) exchanger by sorcin via the interaction of the respective Ca(2+)-binding domains.

Zamparelli C, Macquaide N, Colotti G, Verzili D, Seidler T, Smith GL, Chiancone E - J. Mol. Cell. Cardiol. (2010)

Bottom Line: To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca(2+) due to a mutation at EF3.Downregulation of sorcin decreased and supplementation with wt sorcin (3muM) increased NCX activity in isolated cardiomyocytes.The relative stimulatory effects of the sorcin variants were: W105G>wt sorcin>Sorcin Calcium Binding Domain (SCBD)>W99G>E124A.

View Article: PubMed Central - PubMed

Affiliation: C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, 00185 Rome, Italy. emilia.chiancone@uniroma1.it

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Sequence of Chinese hamster sorcin and structure of the Ca2+-binding domain, SCBD. (A) The amino acids of the N-terminal domain and of SCBD, involved in the interdomain contact according to Ilari et al. [10], are in boldface. The residues mutated in the variants used (W99, W105, F112 and E124) are underlined. (B) left panel. The two monomers in the SCBD dimer are depicted in different colours (light blue, gray). The D helix and the physiologically relevant EF2 and EF3 hands are coloured dark blue; the two tryptophan residues, W99 and W105, are coloured red, Phe112 and Glu124 belonging to EF3 are coloured green. (B) right panel. The gray SCBD monomer is rotated 90° with respect to the dimer presented on the left. The figure was created with PYMOL [42].
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fig1: Sequence of Chinese hamster sorcin and structure of the Ca2+-binding domain, SCBD. (A) The amino acids of the N-terminal domain and of SCBD, involved in the interdomain contact according to Ilari et al. [10], are in boldface. The residues mutated in the variants used (W99, W105, F112 and E124) are underlined. (B) left panel. The two monomers in the SCBD dimer are depicted in different colours (light blue, gray). The D helix and the physiologically relevant EF2 and EF3 hands are coloured dark blue; the two tryptophan residues, W99 and W105, are coloured red, Phe112 and Glu124 belonging to EF3 are coloured green. (B) right panel. The gray SCBD monomer is rotated 90° with respect to the dimer presented on the left. The figure was created with PYMOL [42].

Mentions: Sorcin (soluble resistance-related calcium binding protein) is a 21.6 kDa protein identified in the cytosol of multidrug resistant cells [1,2] that belongs to the penta-EF-hand (PEF) family, a small group of regulatory calcium binding proteins comprising calpain, ALG-2, grancalcin, peflin and PEF1 [3–8]. Sorcin shares the typical structural and functional features of all PEF family members. It has a two-domain architecture, characterized by a flexible and hydrophobic Gly/Pro-rich N-terminal domain and a C-terminal calcium binding domain containing the five EF-hand motifs (Fig. 1), and dimerizes through the unpaired EF5 hand. Like the other PEF proteins, sorcin undergoes a Ca2+-dependent activation that promotes translocation to membranes where interaction with several molecular targets occurs [9]. In turn, these features render sorcin an effective participant in a number of Ca2+-mediated processes. Sorcin activation is induced by Ca2+ binding to the two functionally relevant EF3 and EF2 motifs, that are not paired structurally as in most EF-hand proteins, but are connected by the long and rigid D helix (Fig. 1). An essential step of sorcin activation therefore consists in the transfer of information concerning Ca2+ binding from the site with the highest affinity for the metal, EF3, through the D helix to EF2, and from there to the rest of the molecule. The ensuing conformational change is believed to loosen the hydrophobic and hydrophilic interactions that bring the N- and C-terminal domains together. This renders both domains available for target protein recognition, in particular the D helix residues [10]. It follows that the EF3-D helix-EF2 region should be considered as a tightly coupled functional unit.


Activation of the cardiac Na(+)-Ca(2+) exchanger by sorcin via the interaction of the respective Ca(2+)-binding domains.

Zamparelli C, Macquaide N, Colotti G, Verzili D, Seidler T, Smith GL, Chiancone E - J. Mol. Cell. Cardiol. (2010)

Sequence of Chinese hamster sorcin and structure of the Ca2+-binding domain, SCBD. (A) The amino acids of the N-terminal domain and of SCBD, involved in the interdomain contact according to Ilari et al. [10], are in boldface. The residues mutated in the variants used (W99, W105, F112 and E124) are underlined. (B) left panel. The two monomers in the SCBD dimer are depicted in different colours (light blue, gray). The D helix and the physiologically relevant EF2 and EF3 hands are coloured dark blue; the two tryptophan residues, W99 and W105, are coloured red, Phe112 and Glu124 belonging to EF3 are coloured green. (B) right panel. The gray SCBD monomer is rotated 90° with respect to the dimer presented on the left. The figure was created with PYMOL [42].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2958309&req=5

fig1: Sequence of Chinese hamster sorcin and structure of the Ca2+-binding domain, SCBD. (A) The amino acids of the N-terminal domain and of SCBD, involved in the interdomain contact according to Ilari et al. [10], are in boldface. The residues mutated in the variants used (W99, W105, F112 and E124) are underlined. (B) left panel. The two monomers in the SCBD dimer are depicted in different colours (light blue, gray). The D helix and the physiologically relevant EF2 and EF3 hands are coloured dark blue; the two tryptophan residues, W99 and W105, are coloured red, Phe112 and Glu124 belonging to EF3 are coloured green. (B) right panel. The gray SCBD monomer is rotated 90° with respect to the dimer presented on the left. The figure was created with PYMOL [42].
Mentions: Sorcin (soluble resistance-related calcium binding protein) is a 21.6 kDa protein identified in the cytosol of multidrug resistant cells [1,2] that belongs to the penta-EF-hand (PEF) family, a small group of regulatory calcium binding proteins comprising calpain, ALG-2, grancalcin, peflin and PEF1 [3–8]. Sorcin shares the typical structural and functional features of all PEF family members. It has a two-domain architecture, characterized by a flexible and hydrophobic Gly/Pro-rich N-terminal domain and a C-terminal calcium binding domain containing the five EF-hand motifs (Fig. 1), and dimerizes through the unpaired EF5 hand. Like the other PEF proteins, sorcin undergoes a Ca2+-dependent activation that promotes translocation to membranes where interaction with several molecular targets occurs [9]. In turn, these features render sorcin an effective participant in a number of Ca2+-mediated processes. Sorcin activation is induced by Ca2+ binding to the two functionally relevant EF3 and EF2 motifs, that are not paired structurally as in most EF-hand proteins, but are connected by the long and rigid D helix (Fig. 1). An essential step of sorcin activation therefore consists in the transfer of information concerning Ca2+ binding from the site with the highest affinity for the metal, EF3, through the D helix to EF2, and from there to the rest of the molecule. The ensuing conformational change is believed to loosen the hydrophobic and hydrophilic interactions that bring the N- and C-terminal domains together. This renders both domains available for target protein recognition, in particular the D helix residues [10]. It follows that the EF3-D helix-EF2 region should be considered as a tightly coupled functional unit.

Bottom Line: To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca(2+) due to a mutation at EF3.Downregulation of sorcin decreased and supplementation with wt sorcin (3muM) increased NCX activity in isolated cardiomyocytes.The relative stimulatory effects of the sorcin variants were: W105G>wt sorcin>Sorcin Calcium Binding Domain (SCBD)>W99G>E124A.

View Article: PubMed Central - PubMed

Affiliation: C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, 00185 Rome, Italy. emilia.chiancone@uniroma1.it

Show MeSH
Related in: MedlinePlus