A Ca2+-dependent global conformational change in the 3D structure of phosphorylase kinase obtained from electron microscopy

Phosphorylase kinase (PhK), a Ca2+-dependent regulatory enzyme of the glycogenolytic cascade in skeletal muscle, is a 1.3 MDa hexadecameric oligomer comprising four copies of four distinct subunits, termed alpha, beta, gamma, and delta, the last being endogenous calmodulin. The structures of both nonactivated and Ca2+-activated PhK were determined to elucidate Ca2+-induced structural changes associated with PhK's activation. 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 Angstrom using a 0.5 correlation cutoff, or at 18 Angstrom by the 3sigma (corrected for D-2 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 alpha-, beta, gamma, and delta subunits in the nonactivated 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.