Extended Data Fig. 6. Mutational validation of structural mechanism of neddylated SCF–ARIH1-catalysed ubiquitylation.

a, Close-up of E3–E3act domain with spheres showing locations of strongly defective (red), marginally defective (orange) and hyperactive (green) mutations identified by previous ARIH1 Ala scanning mutagenesis^3,4. Defective mutants map to key CUL1- and RBX1-binding residues, whereas hyperactive mutants map to the site of activating bend-to-kink conformation change within the switch helix. b, Close-up of CUL1-WHB-domain-linked NEDD8 interactions with ARIH1 UBAL domain in TS1, showing locations of strongly defective mutants as red spheres. Mutants in ARIH1 UBAL domain (V123D or F150A) at interface with NEDD8 were previously described⁴. A representative SDS–PAGE gel of experiments testing effects of mutating NEDD8 residues at interface with ARIH1 or with CUL1 in the context of neddylated CUL1–RBX1-activated ubiquitin transfer from UBE2L3 to ARIH1 is shown below. Gel image is representative of independent technical replicates (n = 2). c, Close-up of catalytic elements for TS1 (ubiquitin transfer from E2 UBE2L3 to ARIH1 catalytic cysteine (yellow star)). Red spheres show sites of previously identified strongly defective mutants⁴, or tested on the basis of the structure representing TS1. SDS–PAGE gel (left) shows neddylated CUL1–RBX1-activated ubiquitin transfer from UBE2L3 to ARIH1 or (right) from UBE2L3 via ARIH1 to phosphopeptide substrate derived from cyclin E, testing effects of ARIH1 mutants in the ubiquitin-guided helix preceding the Rcat domain. These residues are markedly remodelled for TS1 and were not tested in the previous ARIH1 Ala scanning mutagenesis study⁴. Gel images are representative of independent technical replicates (n = 2). d, Close-up of catalytic elements for TS2, ubiquitin transfer from ARIH1 to SCF-bound substrate. Spheres indicate sites of mutation defective in achieving substrate targeting configuration. Sites of blue mutations map to region in switch helix contributing to ARIH1 autoinhibition and substrate targeting, and accordingly lead to accumulation of ARIH1~ubiquitin in assays monitoring fluorescent ubiquitin transfer from UBE2L3 to ARIH1 to a neddylated SCF substrate (non-reducing conditions, −DTT (botttom left); reducing conditions, +DTT (bottom right)). Gel images are representative of independent technical replicates (n = 2). e, SDS–PAGE gels monitoring fluorescent ubiquitin transfer to cyclin E phosphopeptide, for ARIH1 triple-glycine mutants probing roles of the N-terminal end of the switch helix of the Ariadne domain. The mutations are upstream of the triple-glycine mutants shown as blue spheres in d. Gel images are representative of independent technical replicates (n = 3). f, SDS–PAGE gels of assays testing whether mutants adopt configuration for ubiquitin transfer from ARIH1 to substrate, probed by reaction with cyclin-E-based TS2 ABP. Gel image is representative of independent technical replicates (n = 2).