Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

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