Evidence for the Location of the Allosteric Activation Switch in the Multisubunit Phosphorylase Kinase Complex from Mass Spectrometric Identification of Chemically Crosslinked Peptides

Phosphorylase kinase (PhK), an (αβγδ)4 complex, regulates glycogenolysis. Its activity, catalyzed by the γ subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory α, β and δ subunits. Activation by phosphorylation is predominantly mediated by the regulatory β subunit, which undergoes a conformational change that is structurally linked with the γ subunit and that is characterized by the ability of a short chemical crosslinker to form β-β dimers. To determine potential regions of interaction of the β and γ subunits, we have used chemical crosslinking and two-hybrid screening. The β and γ subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed “in house” that generates a hypothetical list of crosslinked peptides. A conjugate between β and γ that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of γ (γCRD) and R18 in the N-terminal regulatory region of β (β1–31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1–22 of β inhibited the crosslinking between β and γ, and was itself crosslinked to K303 of γ. In two-hybrid screening, the β1–31 region controlled β subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the β and γ subunits. The sum of our results considered together with previous findings implicates the γCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.