Abstract
The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit. CSN5 alone is inactive due to an auto-inhibited conformation of its catalytic domain. Here we report the molecular basis of CSN5 catalytic domain activation and unravel a molecular hierarchy in CSN deneddylation activity. The association of CSN5 and CSN6 MPN (for Mpr1/Pad1 N-terminal) domains activates its isopeptidase activity. The CSN5/CSN6 module, however, is inefficient in CRL deneddylation, indicating a requirement of further elements in this reaction such as other CSN subunits. A hybrid molecular model of CSN5/CSN6 provides a structural framework to explain these functional observations. Docking this model into a published CSN electron density map and using distance constraints obtained from cross-linking coupled to mass-spectrometry, we find that the C-termini of the CSN subunits could form a helical bundle in the centre of the structure. They likely play a key scaffolding role in the spatial organization of CSN and precise positioning of the dimeric MPN catalytic core.
Highlights
The ubiquitin-proteasome system is implicated in virtually all functions of eukaryotic living cells
The MPN domains of CSN5 and CSN6 form a stable heterodimer Previous structural studies of the Cop9 signalosome (CSN) suggested that CSN5 and
Binding of CSN6DC to a conformationally-relaxed CSN5DC variant, in which the residue arginine 106 of the Ins-1 is substituted by a threonine (CSN5DC,R106T) [19] was probed by Isothermal titration calorimetry (ITC)
Summary
The ubiquitin-proteasome system is implicated in virtually all functions of eukaryotic living cells. As defined in the AMSH (Associated Molecule with the SH3 domain of STAM)-like protein (AMSHLP) [18], the MPN domain of CSN5 contains two insertions, namely Insertion-1 (Ins-1; residues 97–131 in human CSN5) and Insertion-2 (Ins-2; residues 197–219 in human CSN5) These regions usually contribute to the regulation of the isopeptidase activity. Comparison with CSN on synthetic substrates reveals that both CSN and the dimeric MPN module display robust isopeptidase activity towards C-terminal Nedd derivatives, but that CSN is a more efficient deneddylase towards Cullin1-Nedd than the CSN5/ CSN6 MPN complex This suggests that the MPN2 subunit would contribute significantly to the catalytic activity of the human CSN, but efficient substrate recruitment would require additional elements such as other CSN subunits. In addition to the importance of their MPN domains for catalysis, the C-terminal regions of CSN5 and of CSN6 contribute to their anchoring and precise positioning in the CSN assembly
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