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.
Highlights
Neddylated SCF E3 ligases mediate ubiquitylation by the RING domain of RBX1 transiently recruiting a ubiquitin-carrying enzyme from which ubiquitin is transferred to substrates
Ubiquitin is transferred from the ARIH1-bound E2 enzyme UBE2L3 to the catalytic cysteine of ARIH1, and from ARIH1 to the substrate bound to the SCF E3 ligase[4]
Transfer of ubiquitin from the catalytic cysteine of ARIH1 to the substrate bound to the F-box protein is mimicked by simultaneously linking ubiquitin, the catalytic cysteine of ARIH1 and the acceptor site on the cyclin E phosphopeptide. e, Guide to colouring of domains and proteins that participate in E3–E3-mediated ubiquitylation of substrates bound to an F-box protein
Summary
To visualize assemblies that mediate neddylated SCF-dependent ubiquitin transfer from the E2 UBE2L3 to ARIH1 and to a substrate bound to an F-box protein, we obtained cryo-electron microscopy (cryo-EM) data for chemically stable proxies for the two short-lived transition states (TS1 and TS2) and for intervening intermediates (pre-TS1 and post-TS1) The highest-resolution (3.6 Å) map, obtained using the ARIH1 mutant, permitted unambiguous docking of catalytic elements—including ubiquitin linked to the ARIH1 Rcat domain, which was translocated approximately 60 Å from the TS1 active site to confront the F-box-protein-bound substrate (Fig. 1d, Extended Data Fig. 3f). The structural comparison showed the same surfaces of CUL1-linked NEDD8 and the RING domain of RBX1 engaging ARIH1 and UBE2D, albeit in different relative orientations (Extended Data Fig. 5a–d). After a substrate is modified, ubiquitin-free ARIH1 could disengage, allowing the neddylated SCF E3 to use a different ubiquitin-carrying enzyme—presumably one with superior kinetic properties for modifying the particular F-box protein client
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