Abstract
Post-translational modification of proteins with ubiquitin represents a widely used mechanism for cellular regulation. Ubiquitin is activated by an E1 enzyme, transferred to an E2 conjugating enzyme and covalently linked to substrates by one of an estimated 600 E3 ligases (1). RING E3 ligases play a pivotal role in selecting substrates and priming the ubiquitin loaded E2 (E2~Ub) for catalysis (2,3). RING E3 RNF4 is a SUMO targeted ubiquitin ligase (4) with important roles in arsenic therapy for cancer (4,5) and in DNA damage responses (6,7). RNF4 has a RING domain and a substrate recognition domain containing multiple SUMO Interaction Motifs (SIMs) embedded in a region thought to be intrinsically disordered (8). While molecular details of SUMO recognition by the SIMs (8–10) and RING engagement of ubiquitin loaded E2 (3,11–15) have been determined, the mechanism by which SUMO substrate is delivered to the RING to facilitate ubiquitin transfer is an important question to be answered. Here, we show that the intrinsically disordered substrate-recognition domain of RNF4 maintains the SIMs in a compact global architecture that facilitates SUMO binding, while a highly-basic region positions substrate for nucleophilic attack on RING-bound ubiquitin loaded E2. Contrary to our expectation that the substrate recognition domain of RNF4 was completely disordered, distance measurements using single molecule Fluorescence Resonance Energy Transfer (smFRET) and NMR paramagnetic relaxation enhancement (PRE) revealed that it adopts a defined conformation primed for SUMO interaction. Mutational and biochemical analysis indicated that electrostatic interactions involving the highly basic region linking the substrate recognition and RING domains juxtaposed those regions and mediated substrate ubiquitination. Our results 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.
