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The W105G and W99G sorcin mutants demonstrate the role of the D helix in the Ca-dependent interaction with annexin VII and the cardiac ryanodine receptor

Biochemistry, ISSN: 0006-2960, Vol: 45, Issue: 41, Page: 12519-12529
2006
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Article Description

Sorcin, a 21.6 kDa two-domain penta-EF-hand (PEF) protein, when activated by Ca binding, interacts with target proteins in a largely uncharacterized process. The two physiological EF-hands EF3 and EF2 do not belong to a structural pair but are connected by the D helix. To establish whether this helix is instrumental in sorcin activation, two D helix residues were mutated: W105, located near EF3 and involved in a network of interactions, and W99, located near EF2 and facing solvent, were substituted with glycine. Neither mutation alters calcium affinity. The interaction of the W105G and W99G mutants with annexin VII and the cardiac ryanodine receptor (RyR2), requiring the sorcin N-terminal and C-terminal domain, respectively, was studied. Surface plasmon resonance experiments show that binding of annexin VII to W99G occurs at the same Ca concentration as that of the wild type, whereas W105G requires a significantly higher Ca concentration. Ca spark activity of isolated heart cells monitors the sorcin-RyR2 interaction and is unaltered by W105G but is reduced equally by W99G and the wild type. Thus, substitution of W105, via disruption of the network of D helix interactions, affects the capacity of sorcin to recognize and interact with either target at physiological Ca concentrations, while mutation of solvent-facing W99 has little effect. The D helix appears to amplify the localized structural changes that occur at EF3 upon Ca binding and thereby trigger a structural rearrangement that enables interaction of sorcin with its molecular targets. The same activation process may apply to other PEF proteins in view of the D helix conservation. © 2006 American Chemical Society.

Bibliographic Details

Colotti, Gianni; Zamparelli, Carlotta; Verzili, Daniela; Mella, Manuela; Loughrey, Christopher M; Smith, Godfrey L; Chiancone, Emilia

American Chemical Society (ACS)

Biochemistry, Genetics and Molecular Biology

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