The Structure Of Phosphorylase Kinase Holoenzyme At Subnanometer Resolution, Location Of The Catalytic Subunit And The Substrate Glycogen Phosphorylase

Abstract

Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a (GPa) and the stimulation of glycogenolysis. PhK is one of the largest of the protein kinases (MW 1.3 x 106) and contains four copies of four subunits: α,β,γ and δ. Here we present a 9.9Å resolution structure of PhK determined by electron cryo-microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprised of two lobes with 222 symmetry. This 3D structure has allowed us to dock the catalytic γ domain, whose crystal structure is known, to the PhK holoenzyme at a location that is towards the ends of the lobes. The kinase domain is not involved in homo-dimer interactions. We have also determined the structure of PhK decorated with GPb at 18-Å resolution, which shows GPb located at the end of the lobes. Comparison of PhK/GPb complex with the volume of density for the GPb dimer derived from the X-ray model indicates that only one subunit of GPb is localized. Careful examination of the electron microscope images revealed a mixture of large and small PhK particles. In addition to the large particles described above we have determined the reduced size particles at 9.8Å resolution. This structure was consistent with a proteolysed activated form of PhK that had lost the α subunit and possibly the γ subunit.