Rbx1 flexible linker facilitates cullin-RING ligase function before neddylation and after deneddylation

Abstract

In ubiquitination, cullin-RING E3 ubiquitin ligases (CRLs) assist in ubiquitin transfer from ubiquitin-conjugating enzyme E2 to the substrate. Neddylation, which involves NEDD8 transfer from E2 to E3-cullin, stimulates ubiquitination by inducing conformational change in CRLs. However, deneddylation, which removes NEDD8 from cullin, does not suppress ubiquitination in vivo, raising the question of how neddylation/deneddylation exerts its effects. Using molecular-dynamics simulations, we demonstrate that before neddylation occurs, the linker flexibility of Rbx1, a CRL component, leads to conformational changes in CRLs that allow neddylation and initiation of ubiquitination. These large NEDD8-induced conformational changes are retained after deneddylation, allowing both initiation of the ubiquitination process and ubiquitin chain elongation after deneddylation. Furthermore, mutation of lysine, the cullin residue to which NEDD8 covalently attaches, dramatically reduces CRL conformational changes, suggesting that the acceptor lysine allosterically regulates CRLs. Thus, our results imply that neddylation stimulates ubiquitination by CRL conformational control via lysine modification.

Figure 1

Overviews of the CRL E3 ligase machine. A schematic illustration of the Ub-E2-E3-substrate machine shows ubiquitination (A) and neddylation (B). The distances to be bridged are marked. On the left arm, S stands for substrate. The substrate-binding protein has two domains: the substrate-binding domain (SBD) and the Box domain. The adaptor protein mediates between the substrate-binding protein and the cullin. Cullin (Cul) has two domains: Cul NTD and Cul CTD, which contains the Cul CTD 4HB-α/β and Cul CTD WHB subdomains. The Rbx has two domains. The active residues in neddylation (C and K) are marked.

Figure 2

WT conformational changes during the simulation. Snapshots at the beginning of the simulation, the maximum rotations obtained in the two trajectories, as well as the rotation angles for Rbx1 and Cul5 during simulations of the (A and D) closed conformation at 340 K, (B and E) open 1 conformation, and (C and F) open 2 conformation are illustrated. The Cul5 WHB subdomain, Cul5 4HB-α/β subdomains, and Rbx1 are shown in red, gold, and blue, respectively.

Figure 3

Distances between the E2 Cys donor and Cul5 Lys acceptor shorten during simulation of the WT closed conformation at 340 K, allowing the NEDD8 transfer. (A) The snapshot with the smallest distance during the simulation. (B) The snapshot with the largest distance during the simulation. (C) The distance changes as a function of time during the simulation. (D) Normalized histogram for the distribution of the distances. The Cul5 WHB subdomain, Cul5 4HB-α/β subdomains, Rbx1, and E2 are shown in red, gold, blue, and cyan, respectively; Lys is in green, and Cys is in yellow.

Figure 4

Distances between E2 Cys and the substrate fluctuate during the simulations. The snapshot with the largest or shortest distance for the (A and B) WT closed conformation at 340 K, (C and D) open 1 conformation, and (E and F) open 2 conformation. The substrate peptide is in purple, the substrate-binding protein in pink, the adaptor protein in green, the Cul NTD in red, and the Cul CTD, Rbx1, and E2 in gold, blue, and cyan, respectively.

Figure 5

E2-substrate distances during simulations for the closed, open 1, and open 2 conformations. (A) Closed conformation. (B) Open 1 conformation. (C) Open 2 conformation. (D) Normalized histogram for the distribution of the distances.

Figure 6

Cul5 K724R mutants change the E2-substrate distance distribution during the simulation. (A) Closed conformation. (B) Open 1 conformation. (C) Open 2 conformation. (D) Normalized histogram for the distribution of the distances.

Figure 7

Mutation of the Lys cullin acceptor residue to Arg changes the motion correlations between Rbx1 and Cul5. Starting conformations for the covariance maps: (A) closed conformation, (B) open 1 conformation, and (C) open 2 conformation. The WT is shown on the left, the K724R mutant is shown in the middle, and the difference between the WT and mutant is shown on the right. The more red, the stronger the positive correlation; the more blue, the stronger the negative (anti-) correlation. The bar provides the scale. (D) The open 2 K724R conformation showing the domains with the largest correlation change. The Rbx1 RING domain is shown in blue, the Cul5 α/β domain is in orange, and the rest of system is in green. The K724R mutation is shown in red.