A RING E3-substrate complex poised for ubiquitin-like protein transfer: structural insights into cullin-RING ligases

Abstract

How RING E3 ligases mediate E2-to-substrate ubiquitin-like protein (UBL) transfer remains unknown. Here we address how the RING E3 RBX1 positions NEDD8's E2 (UBC12) and substrate (CUL1). We find that existing structures are incompatible with CUL1 NEDD8ylation and report a new conformation of RBX1 that places UBC12 adjacent to CUL1. We propose RING domain rotation as a general mechanism for UBL transfer for the largest family of E3s.

Figure 1

UBC12–RBX1 interactions. (a) WT and mutant UBC12-mediated CUL1 NEDD8ylation in vitro. Mutated residues are orange on UBC12 (otherwise cyan)–RBX1 (pink) model. (b) 2D HSQC [¹⁵N-¹H] spectra of ¹⁵N-UBC12^core in the absence (red) and presence (blue) of equimolar RBX1^RING. A subset of shifted resonances are labeled in the spectra. Residues with chemical shift differences greater than one standard deviation above the mean are labeled and colored orange on UBC12 (otherwise cyan)–RBX1 (pink) model.

Figure 2

Models from prior structures reveal an E2-to-substrate gap. (a) Structural model of CUL1^CTD–RBX1–UBC12^core (colored light green, pink, cyan respectively) complex based on previous CUL1–RBX1 and RING–E2 structures^–,, (Supplementary Fig. 1). CUL1 NEDD8 modification site and UBC12 catalytic cysteine are indicated by blue and yellow spheres respectively. (b) In vitro CUL1 NEDD8ylation assays using disulfide-linked (S~S) and unlinked (SH SH) split’n’coexpress CUL1–RBX1 (ref. 7). Pairs A–C refer to three distinct combinations of engineered cysteines (Supplementary Fig. 2).

Figure 3

Structure of CUL1^CTD–RBX1 in new conformation. (a) One copy of CUL1^CTD (dark green) –RBX1 (purple) from the asymmetric unit. (b) Same as (a), but rotated ~70° in y and ~20° in x. (c) Structural overlay with previous CUL1^CTD (light green) oriented similarly to (a)^,. (d) Structural overlay with previous RBX1 (pink) in same orientation as (b). (e) Structural model of CUL1^CTD–RBX1–UBC12^core (light green, purple, cyan respectively) complex based on the new RBX1 conformation. (f) Bis-Maleimidoethane (BMOE)-crosslinking between engineered cysteine mutants of Ubc12 and CUL1^CTD. Residues mutated to cysteine are numbered on the model (left, view rotated by ~70° and ~40° about the×and z-axes relative to panel e) and products of crosslinking reactions are shown for all possible Ubc12+CUL1–RBX1 combinations (right). Note residue numbering for yeast Ubc12. For reference, Ubc12 catalytic cysteine and CUL1 NEDD8ylation site are shown as sticks. (g) In vitro CUL1 NEDD8ylation for UBC12 mutants at the predicted interface. Mutated residues are shown on the model (left).