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

Abstract

In the tightly regulated glycogenolysis cascade, the breakdown of glycogen to glucose-1-phosphate, phosphorylase kinase (PhK) plays a key role in regulating the activity of glycogen phosphorylase. PhK is a 1.3 MDa hexadecamer, with four copies each of four different subunits (α, β, γ and δ), making the study of its structure challenging. Using hydrogen-deuterium exchange, we have analyzed the regulatory β subunit and the catalytic γ subunit in the context of the intact non-activated PhK complex to study the structure of these subunits and identify regions of surface exposure. Our data suggest that within the non-activated complex the γ subunit assumes an activated conformation and are consistent with a previous docking model of the β subunit within the cryoelectron microscopy envelope of PhK.

Keywords: calmodulin; hydrogen-deuterium exchange; mass spectrometry; molecular modeling; oligomeric proteins; phosphorylase kinase.

Figure 1

Model of β with HDX results. The exchange results for the non‐activated PhK β subunit are mapped onto the theoretical model of β,21 with low levels of exchange in blue, medium levels in green, and high levels of exchange in red, as indicated by the inset. Upper left, 15‐s time point front view, Upper right, 15‐s time point back view, Lower left, 90‐min time point front view, and Lower right, 90‐min time point back view.

Figure 2

β Subunit model domains and subdomains. The domains of the β model as identified by sequence similarity as discussed in the text are separately shown with HDX results at 90 min. Specific regions of interest discussed in the text are labeled.

Figure 3

HDX analysis of the γ subunit catalytic domain. (A) Crystal structure (PDB entry 1PHK) of the catalytic domain of PHK γ,26 with HDX results at 10 min mapped onto the structure (colors as in Fig 1). (B) Structural features of the catalytic domain, including hydrophobic residues composing the R (cyan) and C (slate gray) spines, with the side chains colored (as in Fig. 1). The region of this domain implicated as a subunit contact region40 is indicated by the purple ribbon structure. (C) Magnified view of the stabilizing interaction network between the C‐helix and activation loop of the catalytic cleft.

Figure 4

I‐TASSER threading model of the full‐length γ subunit. (A) Catalytic domain (tan) with CRD (grey) overlaid with the exchange results at 10 min (colors as in Fig 1). (B) Expanded view of side chain contacts between residues from the catalytic domain and the γCRD.