Localization and characterization of the calsequestrin- binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein-protein interaction.
Triadin is an integral membrane protein of the junctional sarcoplasmic reticulum that binds to the high capacity Ca(2+)- binding protein calsequestrin and anchors it to the ryanodine receptor. The lumenal domain of triadin contains multiple repeats of alternating lysine and glutamic acid residues, which have been defined as KEKE motifs and have been proposed to promote protein associations. Here we identified the specific residues of triadin responsible for binding to calsequestrin by mutational analysis of triadin 1, the major cardiac isoform. A series of deletional fusion proteins of triadin 1 was generated, and by using metabolically labeled calsequestrin in filter-overlay assays, the calsequestrin- binding domain of triadin 1 was localized to a single KEKE motif comprised of 25 amino acids. Alanine mutagenesis within this motif demonstrated that the critical amino acids of triadin binding to calsequestrin are the even-numbered residues Lys(210), Lys(212), Glu(214), Lys(216), Gly(218), Gln(220), Lys(222), and Lys(224). Replacement of the odd-numbered residues within this motif by alanine had no effect on calsequestrin binding to triadin. The results suggest a model in which residues 210-224 of triadin form a beta-strand, with the even-numbered residues in the strand interacting with charged residues of calsequestrin, stabilizing a "polar zipper" that links the two proteins together. This small, highly charged beta-strand of triadin may tether calsequestrin to the junctional face membrane, allowing calsequestrin to sequester Ca(2+) in the vicinity of the ryanodine receptor during Ca(2+) uptake and Ca(2+) release.[1]References
- Localization and characterization of the calsequestrin-binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein-protein interaction. Kobayashi, Y.M., Alseikhan, B.A., Jones, L.R. J. Biol. Chem. (2000) [Pubmed]
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