Structural basis of Cullin 2 RING E3 ligase regulation by the COP9 signalosome

Abstract

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.

COP9 signalosome: Discovery, conservation, activity, and function

“…This super‐complex was established upon three pairs of interactions (i.e., CSN2 with CUL4, CSN2, and RBX1 with CSN4, and CSN1 with DDB1), and an induced‐fit mechanism was proposed to account for the principles of CSN activation (Cavadini et al 2016). Moreover, Cryo‐EM structures of the CSN–CRL2~N8 complex and its deneddylated CSN–CRL2 counterpart were very recently elucidated (Faull et al 2019). The detailed procedure of CSN‐mediated deneddylation of CRLs is illustrated in Figure 4.…”

“…( E ) CSN deneddylated the CRL. ( F ) NEDD8 was cleaved from CSN, and CRL was inhibited (adapted from Lingaraju et al 2014; Cavadini et al 2016; Mosadeghi et al 2016; FauII et al 2019). The diagrams are not drawn to scale.…”

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

“…As integrative approaches combining biophysical, structural and biochemical data are more and more required to understand fundamental biological mechanisms, HDX‐MS is certain to shine in the set of biophysical tools accompanying the resolution revolution. A recent study on the Cop9 Signalosome—CRL2 supercomplex provided early evidence of how Cryo‐EM and HDX‐MS can go hand to hand for deciphering mechanistic insights otherwise inaccessible by each technique in isolation …”

Neddylation, an Emerging Mechanism Regulating Cardiac Development and Function

“…Evidence collected from biochemical assays has consistently demonstrated the necessity of reversible cullin neddylation for the assembly and activity of CRLs, as extensively reviewed by others ( Keuss et al, 2019 ). Cryo-electron microscopy analyses of the neddylated CRL1 β TrCP -UBE2D complex and the CSN-CRL2 complex further provide insight into structural mechanisms underlying how NEDD8 activation mediates CRL-catalyzed ubiquitination ( Faull et al, 2019 ; Baek et al, 2020 ). These analyses show that NEDD8 acts as a nexus for conjugating individual cullins and RBX1/RBX2-activated, ubiquitin-charged E2.…”