Figure 4: PCGF helix α3 residues strongly modulate E3 ligase activity as monitored by western blot.

(a) Identities of putative Ub contact residues from helix α3 of the different PCGFs. (b) Auto-ubiquitination catalysed by PCGF4–RING1B mutant complexes in which the two helix α3 residues are taken from the high-activity group PCGFs. ‘P4 M’ denotes the PCGF4 mutant with PCGF5 α3 residues, while ‘P4 KR’ incorporates the K73R mutation (as present in PCGFs 1, 3 and 6). (c) Introduction of human helix α3 residue pairs into Psc of the Psc–Sce complex. Auto-ubiquitination activity tracks with the high or low activity of the corresponding human complex (Fig. 1b). (d) A structural model suggests E3 recognition of ubiquitin lysine residues. Top left: potential interaction between PCGF5 E77 (cyan) and Ub K11 and K33. Bottom: discharge of E2∼Ub (wt or mutants) to lysine catalysed by RING1B–PCGF5. Substrate and product were detected with α-UbcH5c antibody instead of α-Ub to avoid potential differences in detection of different Ub mutants by the latter. Note that the α-UbcH5c antibody used here detects uncharged E2 much more strongly than charged E2. The prominent E2 band at the earliest time point represents only a trace amount of uncharged E2 in the substrate as indicated by Coomassie staining (shown at top center and right).