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    Structural basis for calmodulin-mediated regulation of the ryanodine receptor

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    Author
    Kilpatrick, Adina Maximciuc
    Date
    2008
    Advisor
    MacKenzie, Kevin R.
    Degree
    Doctor of Philosophy
    Abstract
    We investigated the structural basis of calmodulin (CaM)-mediated regulation of the skeletal muscle ryanodine receptor (RyR1), a calcium channel that plays a key role in excitation-contraction coupling in muscle cells. In order to understand the complex interaction of CaM with this receptor, we pursued NMR and X-ray crystallographic studies of CaM/RyR1 peptide complexes both in the presence and in the absence of calcium. We have determined the 2.0 A crystal structure of Ca2+CaM in complex with a 30-residue peptide corresponding to the binding region for CaM on RyR1 (residues 3614 to 3643). The structure reveals that hydrophobic anchor residues in the target arranged in a novel '1-17' spacing allow each calmodulin lobe to interact with the peptide independently. Solution NMR 15N relaxation measurements and residual dipolar couplings confirm the structure of each calmodulin lobe and show that the complex undergoes segmental domain motion. Fluorescence measurements indicate that CaM binds with both domains to the 3614-3643 peptide, whereas if the second anchor is unavailable, CaM can bind without collapsing on the target. The independence of the two lobes of calmodulin offers a structural explanation for how other domains may compete for binding to this region to regulate the channel. NMR studies of these interaction partners indicate that the RyR1 target binds to only the C-lobe of CaM at low calcium concentrations, similar to other ion channels whose activity is modulated by CaM. The conformation of the C-lobe in the calcium-free complex closely resembles the one seen in the crystal structure of the calcium-loaded complex, suggesting that binding of the RyR1 peptide locks the C-domain of CaM in a conformation similar to that of the calcium-loaded protein. Comparison of the CaM/peptide complexes at low and high calcium concentrations provides a model for how CaM interacts with this region of RyR1: the C-lobe is constitutively tethered to the 3614-3643 target and is calcium-loaded even at low, resting calcium levels, whereas high calcium induces the N-lobe to bind to this region. In this way, the N-lobe of CaM acts as a Ca2+ sensor for RyR1 by switching between different binding sites on the receptor.
    Keyword
    Biophysics
    Citation
    Kilpatrick, Adina Maximciuc. "Structural basis for calmodulin-mediated regulation of the ryanodine receptor." (2008) Diss., Rice University. https://hdl.handle.net/1911/22245.
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    Home | FAQ | Contact Us | Privacy Notice | Accessibility Statement
    Managed by the Digital Scholarship Services at Fondren Library, Rice University
    Physical Address: 6100 Main Street, Houston, Texas 77005
    Mailing Address: MS-44, P.O.BOX 1892, Houston, Texas 77251-1892
    Site Map