A model for activation of the hexadecameric phosphorylase kinase complex deduced from zero‐length oxidative crosslinking

Abstract

Phosphorylase kinase (PhK) is a hexadecameric (abcd) 4 enzyme complex that upon activation by phosphorylation stimulates glycogenolysis. Due to its large size (1.3 MDa), elucidating the structural changes associated with the activation of PhK has been challenging, although phosphoactivation has been linked with an increased tendency of the enzyme's regulatory b-subunits to self-associate. Here we report the effect of a peptide mimetic of the phosphoryltable N-termini of b on the selective, zero-length, oxidative crosslinking of these regulatory subunits to form b-b dimers in the nonactivated PhK complex. This peptide stimulated b-b dimer formation when not phosphorylated, but was considerably less effective in its phosphorylated form. Because this peptide mimetic of b competes with its counterpart region in the nonactivated enzyme complex in binding to the catalytic c-subunit, we were able to formulate a structural model for the phosphoactivation of PhK. In this model, the nonactivated state of PhK is maintained by the interaction between the nonphosphorylated N-termini of b and the regulatory C-terminal domains of the c-subunits; phosphorylation of b weakens this interaction, leading to activation of the c-subunits.

Structural characterization of the catalytic γ and regulatory β subunits of phosphorylase kinase in the context of the hexadecameric enzyme complex

“…As discussed above, the structural and functional coupling of the β and γ subunits in the (αβγδ) 4 complex has been characterized by multiple methods . Although crystal structures of the isolated catalytic domain show it to contain all the structural elements of an activated kinase, the HDX results herein also suggest a catalytically competent conformation of γ in the non‐activated PhK complex.…”

“…The key residue targeted by PKA in its activation of PhK and glycogenolysis is Ser‐26 of the β subunit, which lies within the N‐terminal regulatory domain of β, residues 1–31 . This domain appears to mediate an important interaction with the γCRD that keeps the activity of γ from being expressed in non‐activated PhK; upon phosphorylation, the inhibitory function of this region is released, resulting in activation of the PhK complex . Due to the requirement for the β regulatory domain to be exposed and accessible to PKA to allow the phosphorylation of Ser‐26, it was expected that this region should undergo a high level of exchange.…”

“…Also within the PhK complex, the γCRD has been shown to interact with the N‐terminal region of the β subunit near its PKA phosphorylation site, and regions of the β and γ subunits that are proximal within the complex have been shown to be structurally coupled to each other and with PhK activation . Moreover, a model has recently been formulated in which non‐activated PhK is characterized by an interaction between the γCRD and the nonphosphorylated N‐terminus of β, with phosphorylation of the latter weakening that interaction and giving rise to activation of γ …”

Activation of Phosphorylase Kinase by Physiological Temperature

“…A temperature-induced conformational change that leads to activation and involves the N-terminal region of β would be consistent with our recent structural model for activation of PhK by phosphorylation. 30 That report describes activation thusly: “the non-activated state of PhK is maintained by the interaction between the nonphosphorylated N-termini of β and the regulatory C-terminal domains of the γ subunits; phosphorylation of β weakens this interaction, leading to activation of the γ subunits.” Perhaps the transition from 30 to 40 °C similarly weakens the interaction of the N-terminus of β with γ , bringing about activation and a conformational change in β .…”

The regulation of glycogenolysis in the brain

“…tantly large increase in the k cat of its ␥ subunit (40). From results with zero-length oxidative cross-linking and a phosphomimetic peptide of the N terminus of ␤ M (8), a structural model was recently proposed to explain the phospho-activation of mPhK (41). The model posits that the nonactivated conformer of PhK is stabilized by interactions between the ␥ subunit and the nonphosphorylated N terminus of ␤ and that phosphorylation of ␤ weakens this inhibitory interaction, leading to the activation of ␥.…”