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

Abstract

Phosphorylase kinase (PhK), a 1.3-MDa (alphabetagammadelta)(4) hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (alphabetagammadelta)(2) octameric lobes that are oriented back-to-back with overall D(2) symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca(2+) 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 Ca(2+) 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 Ca(2+), the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca(2+)-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 Ca(2+)-activated conformers of PhK are consistent with Ca(2+)-induced conformational changes in both the lobes and the interlobal bridges.

Figure 1.

Small-angle X-ray scattering data from nonactivated and Ca²⁺-activated PhK. (A) Intensity profiles of nonactivated (□) and Ca²⁺-activated (×) PhK and the theoretical scattering profile of each model, respectively (solid curves); symbols are conserved throughout Figure 1. Model scattering intensity profiles were calculated from P(r) curves corresponding to a q range of 0.0054–0.0989 Å⁻¹. The scattering profile of the Ca²⁺-activated conformer was arbitrarily shifted along the ordinate for clarity. (B) Guinier plot of the low-q region of the intensity profiles including the fit to the data (solid lines) in the linear Guinier region corresponding to a q range of 0.01–0.015 Å⁻¹. (C) Guinier plot of intensity profiles corresponding to the ranges used to calculate rod-like features of the holoenzyme reported as apparent R_c1, from 0.0149 to 0.029 Å⁻¹ in q, and apparent R_c2, from 0.0406 to 0.055 Å⁻¹ in q (both linear fits are represented by solid lines).

Figure 2.

Probable vector length distribution functions, P(r) curves, for nonactivated and Ca²⁺-activated PhK. (A) P(r) curve of nonactivated PhK (|) overlaid by the calculated P(r) curve of the nonactivated model (□). (B) P(r) curve of Ca²⁺-activated PhK (|) overlaid by the calculated P(r) curve of the Ca²⁺-activated model (○). (C) Difference length pair distribution function: Effect of Ca²⁺-activation (Consler et al. 1988). Difference P(r) plot relative to nonactivated PhK.

Figure 3.

Models of nonactivated and Ca²⁺-activated PhK. (Row A) Nonactivated models of PhK represented by white spheres for the lobes (ellipsoids) and red spheres for the bridges (cylinders). (Row B) Ca²⁺-activated models of PhK represented by blue-green spheres for the lobes and red spheres for the bridges. (Row C) Nonactivated and Ca²⁺-activated models overlaid for contrast. The colors used for the lobes in rows A and B were transferred to the entire model in row C. (Column 1) Chalice view. (Column 2) Cross view. (Column 3) Butterfly view. (Column 4) Cube view.

Figure 4.

Ca²⁺-induced structural changes in the bridges of nonactivated and Ca²⁺-activated PhK. (Row A) Model structures of nonactivated PhK. (Row B) Model structures of Ca²⁺-activated PhK. (Column 1) Models in the cross orientation with semitransparent lobes to reveal the positions of the red interlobal bridges. (Column 2) Expanded cutout of only the bridge regions of the models in the butterfly orientation.