Solid-state NMR and functional measurements indicate that the conserved tyrosine residues of sarcolipin are involved directly in the inhibition of SERCA1

Eleri Hughes, Jonathan C. Clayton, Ashraf Kitmitto, Mikael Esmann, David A. Middleton

    Research output: Contribution to journalArticlepeer-review

    Abstract

    The transmembrane protein sarcolipin regulates calcium storage in the sarcoplasmic reticulum of skeletal and cardiac muscle cells by modulating the activity of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). The highly conserved C-terminal region (27RSYQY-COOH) of sarcolipin helps to target the protein to the sarcoplasmic reticulum membrane and may also participate in the regulatory interaction between sarcolipin and SERCA. Here we used solid-state NMR measurements of local protein dynamics to illuminate the direct interaction between the Tyr29 and Tyr31 side groups of sarcolipin and skeletal muscle Ca2+-ATPase (SERCA1a) embedded in dioleoylphosphatidylcholine bilayers. Further solid-state NMR experiments together with functional measurements on SERCA1a in the presence of NAc-RSYQY, a peptide representing the conserved region of sarcolipin, suggest that the peptide binds to the same site as the parent protein at the luminal face of SERCA1a, where it reduces Vmax for calcium transport and inhibits ATP hydrolysis with an IC50 of ∼200 μM. The inhibitory effect of NAc-RSYQY is remarkably sequence-specific, with the native aromatic residues being essential for optimal inhibitory activity. This combination of physical and functional measurements highlights the importance of aromatic and polar residues in the C-terminal region of sarcolipin for regulating calcium cycling and muscle contractility. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
    Original languageEnglish
    Pages (from-to)26603-26613
    Number of pages10
    JournalJournal of Biological Chemistry
    Volume282
    Issue number36
    DOIs
    Publication statusPublished - 7 Sept 2007

    Keywords

    • chemistry: Adenosine Triphosphate
    • Animals
    • physiology: Binding Sites
    • chemistry: Calcium
    • physiology: Enzyme Activation
    • Hydrolysis
    • physiology: Ion Transport
    • chemistry: Lipid Bilayers
    • physiology: Muscle Contraction
    • chemistry: Muscle Proteins
    • Nuclear Magnetic Resonance, Biomolecular
    • chemistry: Peptides
    • chemistry: Phosphatidylcholines
    • physiology: Protein Binding
    • chemistry: Proteolipids
    • Rabbits
    • chemistry: Sarcoplasmic Reticulum
    • chemistry: Sarcoplasmic Reticulum Calcium-Transporting ATPases
    • chemistry: Tyrosine

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