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Published online before print March 1, 2005, 10.1110/ps.041124705
Protein Science (2005), 14:1039-1048. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering

Timothy S. Priddy1,2, Brian A. Macdonald3, William T. Heller4, Owen W. Nadeau2, Jill Trewhella3,5 and Gerald M. Carlson2

1 Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri—Kansas City, Kansas City, Missouri 64110, USA2 Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA3 Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA4 Condensed Matter Sciences Division and Center for Structural Molecular Biology, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA

(RECEIVED September 16, 2004; FINAL REVISION December 31, 2004; ACCEPTED December 31, 2004)

Phosphorylase kinase (PhK), a 1.3-MDa ({alpha}{beta}{gamma}{delta})4 hexadecameric complex, is a Ca2+-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched ({alpha}{beta}{gamma}{delta})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 Ca2+-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 Ca2+-induced conformational changes in both the lobes and the interlobal bridges.

Keywords: phosphorylase kinase; small-angle X-ray scattering; modeling; solution structure; Ca2+

Abbreviations: cryoEM, cryoelectron microscopy • dmax, maximum linear dimension • EM, electron microscopy • nsEM, negatively stained electron microscopy • PhK, phosphorylase kinase • Rc, radius of gyration of cross section • Rg, radius of gyration • SAXS, small-angle X-ray scattering • SE-HPLC, size exclusion–high performance liquid chromatography

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041124705.

Reprint requests to: Gerald M. Carlson, Department of Biochemistry and Molecular Biology, Mail Stop 3030, 3901 Rainbow Blvd., Kansas City, KS 66160, USA; e-mail: gcarlson{at}kumc.edu; fax: (913) 588-7440.


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