Abstract
The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.
Highlights
The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub)
While the molecular details of how the Small Ubiquitin-like Modifier (SUMO) Interactions Motifs (SIMs) bind to the SUMO chain substrate and RING domain binds to E2~Ub have been determined, the mechanism by which SUMO chain substrate is delivered to the RING to facilitate ubiquitin transfer remains unresolved
The multidomain protein RING Finger Protein 4 (RNF4) has a RING domain required for dimerization and ubiquitin E3 ligase activity at its C-terminus, while the N-terminal region contains the SIMs (Fig. 1a)
Summary
The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). The N-termius of RNF4, representing about 75% of the whole protein, appears to lack any defined secondary structure[12] It is within this disordered Nterminus that the four tandem SIMs of RNF4 are found, required for binding to its SUMO chain substrate. We show that the N-terminal substrate-recognition domain of RNF4, thought to be intrinsically disordered, maintains the SIMs in a compact global architecture that facilitates SUMO binding, while an arginine-rich motif positions substrate for nucleophilic attack on RING-bound E2~Ub. Contrary to our expectation that the substrate-recognition domain of RNF4 was completely disordered, distance measurements using singlemolecule Förster Resonance Energy Transfer (smFRET) and NMR paramagnetic relaxation enhancement (PRE) reveal that it adopts a defined conformation primed for SUMO interaction. Our results reveal how a defined architecture within a disordered region contributes to substrate ubiquitination by a member of the largest family of ubiquitin E3 ligases
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