Crystal structure and versatile functional roles of the COP9 signalosome subunit 1

Abstract

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) plays key roles in many biological processes, such as repression of photomorphogenesis in plants and protein subcellular localization, DNA-damage response, and NF-κB activation in mammals. It is an evolutionarily conserved eight-protein complex with subunits CSN1 to CSN8 named following the descending order of molecular weights. Here, we report the crystal structure of the largest CSN subunit, CSN1 from Arabidopsis thaliana (atCSN1), which belongs to the Proteasome, COP9 signalosome, Initiation factor 3 (PCI) domain containing CSN subunit family, at 2.7 Å resolution. In contrast to previous predictions and distinct from the PCI-containing 26S proteasome regulatory particle subunit Rpn6 structure, the atCSN1 structure reveals an overall globular fold, with four domains consisting of helical repeat-I, linker helix, helical repeat-II, and the C-terminal PCI domain. Our small-angle X-ray scattering envelope of the CSN1-CSN7 complex agrees with the EM structure of the CSN alone (apo-CSN) and suggests that the PCI end of each molecule may mediate the interaction. Fitting of the CSN1 structure into the CSN-Skp1-Cul1-Fbox (SCF) EM structure shows that the PCI domain of CSN1 situates at the hub of the CSN for interaction with several other subunits whereas the linker helix and helical repeat-II of CSN1 contacts SCF using a conserved surface patch. Furthermore, we show that, in human, the C-terminal tail of CSN1, a segment not included in our crystal structure, interacts with IκBα in the NF-κB pathway. Therefore, the CSN complex uses multiple mechanisms to hinder NF-κB activation, a principle likely to hold true for its regulation of many other targets and pathways.T he constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a more than 300-kDa complex that was first identified as a negative regulator of Constitutive Photomorphogenesis (COP) in plants (1, 2). In the subsequent years, the highly conserved protein complex was also found in fungi (3, 4), Caenorhabditis elegans (5), Drosophila melanogaster (6), and mammals (7,8). The most studied function of the CSN complex in eukaryotes is the regulation of protein degradation through two pathways, deneddylation (9-11) and deubiquitination (12, 13). In the deneddylation pathway, the CSN complex can influence the cullin-RING ligase activity by removing Nedd8, a ubiquitin-like protein, from a cullin (9, 14). On the other hand, the CSN complex can also suppress cullin activity through recruitment of the deubiquitination enzyme USP15 (12) or Ubp12p, the Schizosaccharomyces pombe ortholog of human USP15 (13). Other functions of the CSN complex identified in mammalian cells include regulating the phosphorylation of ubiquitin-proteasome pathway substrates through CSN-associated kinases (7,(15)(16)(17)(18). Overall, the CSN complex appears to be a key player in protein subcellular localization (19,20), DNA-damage response (21), NF-κB activation (22), development, and cell cycle control (23,24). Thus, the functions ...

Structural insights of a hormone sensitive lipase homologue Est22

“…The supernatant was purified using a His Trap TM HP column (GE Healthcare) and was eluted in a buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 5% Glycerol, pH 8.0). The protein was further purified by gel filtration using a Superdex 200 16/600 column (GE Healthcare) in a gel filtration buffer (20 mM Tris-HCl, 100 mM NaCl, 2 mM DTT, pH 7.4) 26 . The purity of the Est22 protein was confirmed by SDS-PAGE and concentration was determined by the method of Bradford with bovine serum albumin (BSA) as a standard.…”

Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex

“…S3) (31,(45)(46)(47)(48)(49)(50)(51)(52)(53)(54). In addition, structurally defined remote homologs (PDB codes 2QX5 (chain B) and 4LCT (chain A)) (55,56) were detected for the C-terminal domain in Nup85 by HHpred (57, 58) (supplemental Fig. S3).…”

Diversity of COP9 signalosome structures and functional consequences

“…Whereas USP15 seems to protect components of CRLs [37,41], interaction with Den1 is accompanied with Den1 destabilization [40]. Moreover, CSN-associated USP15 and USP48 control CRL substrates IjBa and RelA [39,42,43]. In collaboration with the UPS the CSN is involved in cell cycle progression [14], DNA repair [14,44], cell differentiation [45][46][47][48], development [15,45,47] as well as in tumorigenesis [49,50].…”