“Three-Dimensional Structures of Non-activated and Ca2+-Activated Phosphorylase Kinase from Electron Microscopy Reveal a Ca2+-Dependent Global Conformational Change”
Owen Nadeau – University of Kansas Medical Center
Phosphorylase kinase (PhK), a regulatory enzyme of the glycogenolytic cascade, is a 1.3 megadalton hexadecameric oligomer comprising four copies of four distinct subunits, termed a, b, g and d, the last being endogenous calmodulin. Activation of PhK by Ca2+ in skeletal muscle directly links energy production with muscle contraction. The structures of both non-activated and Ca2+-activated PhK were determined to elucidate Ca2+-induced structural changes associated with the coordinate regulation of glycogenolysis and muscle contraction. Minimal dose electron micrographs of negatively stained PhK in its non-activated and Ca2+-activated conformers revealed particles in a multitude of orientations. A simple model was used to orient the individual images for three-dimensional reconstruction, followed by multiple rounds of refinement. Reconstructions of both conformers of the kinase, each including over 11,000 particles, yielded bridged, bilobal structures with resolutions estimated by Fourier Shell Correlation at 24, using a 0.5 correlation cut-off, or at 18 by the 3s (corrected for D2 symmetry) threshold curve. Extensive Ca2+-induced structural changes were observed in regions encompassing both the lobes and bridges, consistent with changes in subunit interactions upon activation. The relative placement of the a, b, g and d subunits in the non-activated three-dimensional structure, relying upon previous two-dimensional localizations, is in agreement with the known effects of Ca2+ on subunit conformations and interactions in the PhK complex. These results suggest that a global conformational change occurs in response to the binding of Ca2+ by PhK’s intrinsic calmodulin (d) subunit. Ongoing studies of PhK employing cryo-electron microscopy will also be discussed.