Ubiquitin ligation to F-box protein targets by SCF–RBR E3–E3 super-assembly

Abstract

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin–RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family—which together account for nearly half of all ubiquitin ligases in humans—form E3–E3 super-assemblies3–7. Here, by studying CRLs in the SKP1–CUL1–F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3–E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3–E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3–E3 super-assembly may therefore underlie widespread ubiquitylation.

An E3 ligase guide to the galaxy of small-molecule-induced protein degradation

“…Most subunits of the APC/C, for example, are present throughout the cell cycle, but a combination of inhibitors and posttranslational modifications ensures that this E3 ligase ubiquitylates its targets during mitosis and in the subsequent G1 phase (Watson et al, 2019). The same applies to RBR family E3 ligases that often exist in autoinhibited conformations that are overcome by specific partners, including other E3 ligases or phosphorylated ubiquitin (Duda et al, 2013;Horn-Ghetko et al, 2021;Lechtenberg et al, 2016;Wauer et al, 2015). In addition, as shown for Cullin RING ligases, even expressed substrate adaptors do not form an active E3 ligase unless they have recognized their target and are allowed to engage the cullin scaffold in a process promoted by the exchange factor CAND1 (Liu et al, 2018;Pierce et al, 2013;Reichermeier et al, 2020).…”

Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2

“…Cyclin F was combined with neddylated Cul1, Skp1, substrate, and ubiquitin. The human homologue of ariadne ( ARIH1) is a RING-Between-RING (RBR) E3 ligase that has been shown to work with the E2 UBCH7 and other cullin ring E3 ligases, including the SCF, to ubiquitinate substrates (Horn-Ghetko et al, 2021;Scott et al, 2016). Therefore, ARIH1/UBCH7 and the chainelongating E2 CDC34b were used as the ubiquitin transfer module.…”

Med Chem Remote: The Frontiers in Medicinal Chemistry 2021

“…As last speaker of this session, Klaus Rumpel from Boehringer Ingelheim (BI) presented findings from BIs BCL6 inhibitor program, where he and his colleagues surprisingly identified BCL6‐degrading compounds [5] . These compounds have been very influential in the field and most recently where shown to induce BCL6 polymerization ultimately leading to degradation [6] . Taken together, contributions by Thomä, Schulman and Rumpel were highly stimulating for the audience and their findings will be important to the vibrant field of targeted protein degradation.…”