Fig. 7: Overall structure of FL PRT1 and RING dimerization.

a SEC-SAXS envelope model of PRT1. PRT1 has an L-shaped structure. The upper part of the model corresponds to the C-terminus, where the ZZ domain is located. The dimeric RING domain is positioned at the curved body. The ZZ domain is colored cyan, and the AlphaFold2 model of the dimeric RING domain is colored blue (RING1) and salmon (RING2) in the atomic models fitted into the SAXS molecular envelope. b RING1 and RING2 heterodimer formation. This model was obtained using AlphaFold, and the mutated residues (4 L/S: L79S/L82S in RING1 and L246S/L249S in RING2) for disrupting dimers are shown as yellow sticks. c Peak migration profile for characterizing tandem RING domains. RING2^WT co-migrates with RING1^WT, whereas RING2^2L/S (L246S/L249S double mutant) behaves independently. Both species in peak 1 are too low a concentration to calculate experimental molecular weights. The detailed SEC-MALS analysis for peak 2 is shown in Supplementary Fig. 14c. UV absorption was measured at a 280-nm wavelength. The results for the RING1^WT + RING2^WT mixture are represented by a yellow line, and those for RING1^WT + RING2^2L/S by a blue line. Schematic oligomeric states of RING domains are shown below the UV profile. d Corresponding gel images for the fractions in (c). RING1^WT and RING2^WT are shown at 20 and 10 kDa, respectively. We observed consistent results at least three times. e Ubiquitylation assay of PRT1^WT and 4 L/S mutant. When the dimeric interface of RING domains was disrupted, the ubiquitylation activity of PRT1 was lost. Each of the samples was incubated at 30 °C for 3 h. Ctrl: No PRT1. The ubiquitylation assays were performed at least three times. f FRET-based Ub discharge assay. The E2~Ub discharge activity of the 4 L/S mutant was decreased by 10-fold compared to the WT. The normalized FRET data was analyzed by one-phase decay analysis with GraphPad Prism 5. The data are represented by mean ± SEM from three independent experiments.