title: The structure of phosphorylase kinase holoenzyme at 9.9 angstroms resolution and 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. 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.
authors.affiliation: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU Oxford, UK. catherine.venien@bioch.ox.ac.uk
authors.forename: Catherine
authors.initials: C
authors.lastname: Vénien-Bryan
authors.forename: Slavica
authors.initials: S
authors.lastname: Jonic
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Skamnaki
authors.forename: Nick
authors.initials: N
authors.lastname: Brown
authors.forename: Nicolas
authors.initials: N
authors.lastname: Bischler
authors.forename: Nikos G
authors.initials: NG
authors.lastname: Oikonomakos
authors.forename: Nicolas
authors.initials: N
authors.lastname: Boisset
authors.forename: Louise N
authors.initials: LN
authors.lastname: Johnson
mesh_terms: 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
chemical_list: D024981:Glycogen Phosphorylase; D010764:Phosphorylase Kinase