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Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)(4) complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)(2) lobes joined with D(2) symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)(4) complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)(4) kinase core, because a β(4) subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.

Original publication

DOI

10.1074/jbc.M112.412874

Type

Journal article

Journal

J Biol Chem

Publication Date

26/10/2012

Volume

287

Pages

36651 - 36661

Keywords

Amino Acid Sequence, Animals, Cross-Linking Reagents, Dinitrofluorobenzene, Molecular Docking Simulation, Peptide Fragments, Phosphorylase Kinase, Protein Binding, Protein Interaction Domains and Motifs, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Subunits, Rabbits, Tandem Mass Spectrometry