Abstract
The COP9-Signalosome (CSN) regulates cullin-RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network.
Highlights
Cullin–RING ubiquitin ligases comprise one of the largest families of regulatory enzymes in eukaryotic cells (Deshaies and Joazeiro, 2009)
This suggests a model wherein a cullin–RING ubiquitin ligase (CRL) complex has a higher probability of being conjugated to Nedd8 as long as it is bound to substrate
To gain detailed insights into the molecular determinants underlying activation of CSN, we performed cryo electron microscopy and single particle analysis of CSN5H138A in complex with neddylated SCFSkp2/Cks1 (the sample is described in Enchev et al, (2012) (Figure 1A, Figure 1—figure supplement 1, Figure 1—figure supplement 2A)
Summary
Cullin–RING ubiquitin ligases comprise one of the largest families of regulatory enzymes in eukaryotic cells (Deshaies and Joazeiro, 2009). Structural analysis suggests that a CRL ubiquitination substrate bound to a substrate receptor sterically prevents concurrent binding of CSN (Enchev et al, 2012; Fischer et al, 2011) This suggests a model wherein a CRL complex has a higher probability of being conjugated to Nedd (and of being shielded from Cand1) as long as it is bound to substrate. Comparison of the structure of free CSN to the structure of a catalytically-dead mutant CSN bound to Nedd8-conjugated SCFSkp determined by negative stain electron microscopy (Enchev et al, 2012) implied that binding of substrate to CSN may induce several conformational changes in the latter, including movement of the N-terminal domains (NTD) of Csn and Csn towards the cullin The latter movement, in turn, might be further propagated to the Csn5/6 module (Lingaraju et al, 2014). At present, the mechanism of how CSN is switched on and off and the significance of this switching behavior remain unknown
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