The Structure of Phosphorylase Kinase Holoenzyme at 9.9 Å Resolution and Location of the Catalytic Subunit and the Substrate Glycogen Phosphorylase

• Published in: Structure (London) 2009-01, Vol.17 (1), p.117-127
• Main Authors: Vénien-Bryan, Catherine, Jonic, Slavica, Skamnaki, Vasiliki, Brown, Nick, Bischler, Nicolas, Oikonomakos, Nikos G., Boisset, Nicolas, Johnson, Louise N.
• Format: Article
• Language: English
• Subjects: Biochemistry, Molecular Biology; Life Sciences; PROTEINS; SIGNALING
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2008.10.013

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. We present a 9.9 Å resolution structure of PhK heterotetramer (αβγδ)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic γ subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 Å resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 Å resolution was consistent with a proteolysed activated form of PhK that had lost the α subunits and possibly the γ subunits.