Fig. 5: UFC1 possesses an additional oxyanion hole that is TAK-independent.

a Super-positioning UFC1 C116E (blue) onto the structure of Ubc1 charged with Ub (pink) (PDB 1fxt). For simplicity, Ubc1 is omitted, with only its active site C88, and the linked Ub is shown. The Ub G76 carbonyl adopts a different conformation, relative to the UFC1 E116 side chain and is oriented toward the L117 and T118 C-alpha atoms. b A representative western blot showing the levels of mono-ufmylated WT UFC1 and UFC1 T106I in an in vitro ufmylation assay without E3 (See Supplementary Fig. 6b for protein loading controls). c The band corresponding to mono-ufmylated UFC1 was quantified for the T106I mutant and was normalized against mono-ufmylated UFC1 formed in WT protein (data represented as mean ± SD, n = 4 independent experiments). The difference was significant (p < 0.001, one‐tailed unpaired Student’s t‐test). d and e. A western blot showing the effect of WT UFC1 and the T106I mutant on in vitro ufmylation of UFC1 C116A. Blots are shown with anti-FLAG antibody as UFM1 has a FLAG-tag (d) and with anti-His antibody against Trx-UFC1 C116A that has a His-tag (e) (for protein loading controls see Supplementary Fig. 6c, d respectively). f Western blot of an in vitro ufmylation assay in the absence of E3, comparing the levels of mono-ufmylated UFC1 in the indicated mutants (see Supplementary Fig. 6e for protein loading controls). g Western blot of an in vitro ufmylation assay in the presence and absence of DDRGK1-UFL1. Only UFC1 WT and K122R in the presence of DDRGK1-UFL1 show an additional band of ~40 kDa (see Supplementary Fig. 6f for protein loading controls). Data in d, g and e, f are representative of two and three independent experiments, respectively. Source data are provided as a Source Data file.