Final Report Summary - CSN (Multimodal Regulation of Cullin-RING Ligases by CSN)
Cullin-RING ubiquitin ligases (CRLs) play key roles in virtually every aspect of eukaryotic cellular metabolism in health and disease and are prominent regulators of cell cycle progression (Deshaies and Joazeiro, 2009). The modular CRL architecture is the key to their ubiquitous but highly specific functions. As exemplified by the archetypical CRL, Skp1-Cullin1-F-box (SCF), one of seven different cullin scaffolds binds a RING-finger protein, e.g. Rbx1, which itself associates with a ubiquitin-loaded ubiquitin-conjugation E2 enzyme. Substrate specificity is conferred by a substrate receptor (SR) module located at the opposite end of the. SRs are themselves organized in a modular and highly variable manner, allowing recognition of numerous substrates by the same CRL catalytic core. For SCF alone there are over 60 different SRs, usually given as a superscript (e.g. SCFFbw7). Nedd8 conjugation (neddylation) to cullins triggers a major conformational rearrangement, which significantly up-regulates CRL-mediated ubiquitination and is incompatible with binding of the CRL-regulator CAND1 (Duda et al., 2008; Saha and Deshaies, 2008). While much progress has been made in understanding cullin neddylation (reviewed by ((Rabut and Peter, 2008) and (Duda et al., 2011)), little was known about Nedd8-deconjugation and inhibitory CRL regulation mechanisms.
The COP9 Signalosome (CSN) deneddylates CRLs, but this complex molecular machine is almost three times larger than its cullin substrate, hinting at yet unknown functions and regulation mechanisms. CSN comprises eight subunits that together unleash the metalloprotease activity of its Csn5 subunit by an unknown mechanism (Sharon et al., 2009). Even more perplexing, despite suppressing CRL activity in vitro, CSN is necessary for CRL function in vivo (Cope and Deshaies, 2003). This is known as the ‘CSN paradox’. Although CSN associates with fully assembled CRLs in cells even after deneddylation (Bennett et al., 2010), structural understanding of the interaction and a coherent mechanistic explanation were lacking.
Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCFSkp2/Cks1 and SCFFbw7 and found that CSN occludes both SCF functional sites - the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and it inhibits SCF-catalyzed ubiquitin chain forma- tion independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state and inhibits a catalytically competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination.
The COP9 Signalosome (CSN) deneddylates CRLs, but this complex molecular machine is almost three times larger than its cullin substrate, hinting at yet unknown functions and regulation mechanisms. CSN comprises eight subunits that together unleash the metalloprotease activity of its Csn5 subunit by an unknown mechanism (Sharon et al., 2009). Even more perplexing, despite suppressing CRL activity in vitro, CSN is necessary for CRL function in vivo (Cope and Deshaies, 2003). This is known as the ‘CSN paradox’. Although CSN associates with fully assembled CRLs in cells even after deneddylation (Bennett et al., 2010), structural understanding of the interaction and a coherent mechanistic explanation were lacking.
Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCFSkp2/Cks1 and SCFFbw7 and found that CSN occludes both SCF functional sites - the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and it inhibits SCF-catalyzed ubiquitin chain forma- tion independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state and inhibits a catalytically competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination.