Following Ariadne's thread: a new perspective on RBR ubiquitin ligases

Abstract

Ubiquitin signaling pathways rely on E3 ligases for effecting the final transfer of ubiquitin from E2 ubiquitin conjugating enzymes to a protein target. Here we re-evaluate the hybrid RING/HECT mechanism used by the E3 family RING-between-RINGs (RBRs) to transfer ubiquitin to substrates. We place RBRs into the context of current knowledge of HECT and RING E3s. Although not as abundant as the other types of E3s (there are only slightly more than a dozen RBR E3s in the human genome), RBRs are conserved in all eukaryotes and play important roles in biology. Re-evaluation of RBR ligases as RING/HECT E3s provokes new questions and challenges the field.

Figure 1

RING and HECT-type mechanisms of ubiquitin transfer. (a) On the left, a RING E3 ligase (blue) is shown bound to a ubiquitin-conjugated E2, from which the ubiquitin is transferred to a lysine on the substrate. On the right, a HECT E3 ligase (orange) is shown bound to a ubiquitin-conjugated E2, from which ubiquitin is first transferred to the active-site cysteine of the E3, and is then transferred to a lysine on the substrate bound to the E3 (lower panel). (b) Proposed mechanism for RBR ubiquitin transfer. RBR ligases combine features of both RING- and HECT-type ligases. The ubiquitin-conjugated E2 binds to the RING1 domain of the RBR E3 ligase. The ubiquitin is then transferred from the E2 to the E3 RING2 domain from which it is transferred to the substrate.

Figure 2

RING1 of RBRs maintains features characteristic of canonical RINGs. (a) Structures of RING domains are displayed with Zn²⁺ coordinating residues as yellow sticks and Zn²⁺ ions displayed as grey spheres. A conserved hydrophobic residue important for E2 binding is shown as orange sticks. The structures are (from left to right) the E3 ligase CNOT4 (blue) bound to the E2 UbcH5b (purple) (PDB ID 1UR6) (the E2 active site is shown as yellow spheres); the heterodimeric RING E3 ligase BRCA1 (blue)/BARD1 (green) (PDB ID 1JM7); TRAF6 (PDB ID 3HCT); RING1 of the RBR E3 RNF144 (PDB ID 1WIM). (b) Multiple sequence alignment of the RING domains of CNOT4, BRCA1, TRAF6, and RNF144. Coloring in the multiple sequence alignment corresponds with the colors in the structures, highlighting residues important for Zn²⁺ coordination and E2 binding. Sequences were aligned using CLUSTALW and manually adjusted based on structure [44].

Figure 3

Conservation of the IBR domain. (a) Structures of IBR domains solved to date from RNF31 (left) (PDB ID 2CT7) and Parkin (right) (PDB ID 2JMO). (b) Multiple sequence alignment of the IBR domain from human RBR ligases. Residue numbers are shown at the beginning of the alignment. Sequences were aligned using CLUSTALW [44]. Swiss-Prot numbers for sequences used in multiple sequence alignments are as follows: Cullin-9: Q81WT3, Parkin: O60260, ANKIB1: Q9P2G1, ARIH1: Q9Y4X5, ARIH2: O95376, RBCK1: Q9BYM8, RNF144A: P50876, RNF144B:Q7Z419, RNF19A:Q9NV58, RNF19B: Q6ZMZ0, RNF216: Q9NWF9, RNF14: Q9UBS8, and RNF31: Q96EP0.

Figure 4

Comparison of HHARI RING2 with Parkin RING2. (a) Multiple sequence alignment of HHARI RING2 and Parkin RING2. Zn²⁺-liganding residues determined structurally for HHARI RING2 are denoted in yellow. Potential additional Zn²⁺ coordinating residues in Parkin as proposed by Rankin et al. [34] are highlighted in red. The active site cysteine is denoted by a double dagger. (b) The structure of HHARI RING2 displays Zn²⁺-liganding residues as yellow sticks. The active site cysteine is shown as orange sticks (PDB ID 1WD2). Sequences were aligned using CLUSTALW [44].