Fig. 4: K108 side chain is essential for ufmylation activity.

a Western blot showing the effect of K108 mutants of UFC1 (K108A, K108M, K108R) on in vitro ufmylation assay (see Supplementary Fig. 5a for protein loading control). b Western blot showing the effect of K108A, K108M, and K108R mutants on RPL26 ufmylation. Ufmylation reactions were performed in the presence of the 60S ribosomes and the above mutants. (Supplementary Fig. 5b for protein loading control). Data in a, b are representative of three independent experiments. Source data are provided as a Source Data file. c B-factor analysis for the C-alpha atom in position 108 for WT UFC1 (2z6p) and the indicated mutants. d The UBC9 cleft next to the active site is shown in the structure of UBC9-SUMO2-RANGAP1 (PDB 5d2m). This cleft facilitates the entry of the lysine from one end and the C-terminus of SUMO2 from the other. UBC9 is depicted in yellow using a surface representation. K524 of RANGAP1 is colored green and is linked to the C-terminal glycine of SUMO2 (gray). e Superposition of UFC1 (blue) and UBC9 (yellow) from the structure, depicted in panel d, shows that the side chain of K108 lines the top of the cleft. In UBC9, this region is formed by a loop that is absent in UFC1. Therefore, the K108 side chain is critical for generating a proper cavity to accommodate the attacking lysine and the C-terminus of UFM1. f As in panel e, but for simplicity, UBC9 is not shown. UFC1 is displayed in surface representation, allowing better visualization of the cleft adjacent to the active site and the role of K108 in shaping it. g and h Superposition of WT UFC1 with the UFC1 mutants K108R (g) and K108M (h). The residue at position 108 is shown in the stick representation. In both mutants, the residue enters the cleft and narrows its size. i Superposition of UFC1 K108A/C116E (shown in green) with WT UFC1. K108 of WT UFC1 is shown in stick representation colored blue. The short side chain of A108 prevents the formation of the top of the cleft.