The structure of phosphorylase kinase holoenzyme at 9.9 angstroms resolution and location of the catalytic subunit and the substrate glycogen phosphorylase.
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 angstroms resolution structure of PhK heterotetramer (alphabetagammadelta)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 gamma 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 angstroms 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 angstroms resolution was consistent with a proteolysed activated form of PhK that had lost the alpha subunits and possibly the gamma subunits.
Vénien-Bryan
Catherine
C
Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU Oxford, UK. catherine.venien@bioch.ox.ac.uk
Jonic
Slavica
S
Skamnaki
Vasiliki
V
Brown
Nick
N
Bischler
Nicolas
N
Oikonomakos
Nikos G
NG
Boisset
Nicolas
N
Johnson
Louise N
LN
D020134:Catalytic Domain; D002850:Chromatography, Gel; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D024981:Glycogen Phosphorylase / Q000378:metabolism*; D008958:Models, Molecular; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
D024981:Glycogen Phosphorylase; D010764:Phosphorylase Kinase