Ca²⁺-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering

Phosphorylase kinase (PhK), a 1.3-MDa (αβγδ) ₄ hexadecameric complex, is a Ca²⁺-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (αβγδ)₂ octameric lobes that are oriented back-to-back with overall D₂ symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca²⁺ by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca²⁺ in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca ²⁺-activated PhK. Although the overall dimensions of the complex were not affected by Ca²⁺, the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca²⁺-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca²⁺-activated conformers of PhK are consistent with Ca ²⁺-induced conformational changes in both the lobes and the interlobal bridges.