title: Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes.
abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with the 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation from erythrocytes.
authors: Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors: Beate
authors: B
authors: Rockel
authors: Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
authors: Tilo
authors: T
authors: Schmaler
authors: Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
authors: Xiaohua
authors: X
authors: Huang
authors: Division of Molecular Biology, Department of General, Visceral, Vascular and Thoracic Surgery, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. Electronic address: Wolfgang.dubiel@charite.de.
authors: Wolfgang
authors: W
authors: Dubiel
mesh_terms: D000818:Animals; D000075686:COP9 Signalosome Complex; D044842:Cullin Proteins / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D051379:Mice; D008854:Microscopy, Electron; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D000075687:NEDD8 Protein; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: C100318:Cullin 1; D044842:Cullin Proteins; D046912:Multiprotein Complexes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C513172:Nedd8 protein, mouse; D009842:Oligopeptides; D014452:Ubiquitins; C085706:FLAG peptide; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex
keywords: 3D-reconstruction; COP9 signalosome; Cryo-electron microscopy; Deneddylation; Lid; Single-particle analysis