A mechanistic view of cullin neddylation: allosteric conformational control of E3-ring ligases

Abstract

Cullin-RING E3 ligases (CRLs), which constitute a superfamily of RING type E3 ligases, regulate ~20% of protein degradation by the ubiquitin-proteasome system in the cells. Cullin neddylation activates CRLs by urging a conformational transition in Cullin C-terminal domain and exposes the Rbx1 RING E3 ligase to facilitate ubiquitination. CRLs are over-activated by Cullin neddylation in myriad of human cancers; thereby the inhibition mechanism of Cullin neddylation is of significant interest for generating novel cancer therapies. To explore how the signals of Cullin neddylation are transmitted through the CRLs to control the Rbx1 conformations, we performed molecular dynamics (MD) simulations combined with Gaussian Network Model (GNM) analysis on the closed-unbound and open-bound to Nedd8 states of Cul5-Rbx1 E3 ligases. Our results display multiple Rbx1 RING domain conformations with neddylation of Cul5 where the rigid dynamics of the unbound-to-Nedd8 state CRLs is abrogated. The Rbx1 RING domain conformations of the neddylated CRLs, which are in agreement with the proposed neddylated state Rbx1 conformations for effective ubiquitin transfer, arise as a result of coupled dynamics between Nedd8 and Rbx1. A dynamic hinge plane of Cul5 hinges at R495-M496, N704-E705 and Rbx1 hinges at D51-L52, T69-V70 coordinates allosteric communication between Nedd8 and Rbx1 on the CRL structure along an H-bond between Cul5 R538, R569 and Rbx1 E67 that restricts the Rbx1 conformational space. Nedd8 predisposes Rbx1 together with Cul5 for an effective ubiquitin transfer by a plausible mechanism explaining the allosteric conformational control of Rbx1. We propose that selective interruptions in the neddylation pathway to disrupt the dynamic hinge plane coordination could shed a light on the allosteric drug design studies on Cullin neddylation inhibition.