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Structural Mechanism For Small-molecule Activation Of The Serca Calcium Pump In Muscular Dystrophy: 2635 Board #1 June 1 3

PURPOSE: We have characterized a small-molecule activator of SERCA, the sarcoplasmic reticulum calcium transporting ATPase in muscle. A hallmark of muscular dystrophy (MD) is decreased sarcolemma stability and increased calcium influx, leading to myocyte damage and death. Increasing SERCA activity by gene therapy reverses MD in cell culture and animal models. We continue the characterization of recently-discovered small-molecule activators (SMA) of SERCA by the Thomas lab, since their binding site on SERCA was unknown. METHODS: Small-molecule activation of SERCA was characterized by three techniques: enzyme assays, fluorescence spectroscopy, and x-ray crystallography, with particular focus on the potent SMA1163. RESULTS: ATPase assays indicate that SMA1163 stimulates SERCA activity by 36 ± 11%, with an EC 50 of 3 ± 1 μM (n=6). SMA1163 activated both ATPase activity and calcium transport by SERCA. Tryptophan (TRP) residues of SERCA exhibit a potent, dose-dependent fluorescence quenching by SMA1163, with an EC 50 = 2 ± 1 μM, indicating a transmembrane binding site of SMA1163. Fluorescence of a site-directed fluorescein probe (FITC) identifies kinetic transitions resulting from SMA activation, including enhancement of calcium binding and phosphate release. Conformation-specific proteolytic cleavage and intramolecular glutaraldehyde cross-linking were used to determine the effect on SERCA headpiece structure, indicating that SMA1163 retains normal ligand-binding effects. An atomic structure of SERCA+SMA1163, determined by x-ray crystallography, indicates a transmembrane domain binding site for SMA1163 on SERCA, consistent with TRP fluorescence. Furthermore, the binding site of SMA1163 is located on the energy transduction segment of SERCA, consistent with the kinetic mechanisms of activation detected by FITC fluorescence. CONCLUSIONS: We propose that SMA1163 may be a useful activator of SERCA calcium transport to help alleviate MD, as an alternative approach to SERCA gene therapy.