Fig. 6: Structural modeling suggests C-alpha hydrogen bonding in E2 enzymes possessing an HPN motif.

a Super-positioning of His and Asn in the HPN motif of different E2 enzymes, specifically, UBC9 (PDB 1a3s), E2-25K (PDB 2bep), UBC7 (PDB 2cyx), Rad6 (PDB 2yb6), and E2S (PDB 1zdn), shows a conserved hydrogen bond between the side chain of His and the backbone nitrogen of Asn. b–d Super-positioning of the crystal structures of UBC9 C93E, Ubc13 C87E, and UBCH5B C85E onto the corresponding WT structure of each of these E2 mutants charged with Ub/UBL (PDBs; 5d2m, 2gmi and 3a33; Ub/UBL is omitted from the figure for simplicity). The conformation of UFC1 E116 (side chain shown in stick representation (blue), with van der Waals surface) super-imposed onto the corresponding Glu in each E2 enzyme. e Western blot showing the effect of UFC1 (HPN) on ufmylation of ribosomal protein RPL26. Ufmylation reactions were performed in the presence of the 60S ribosomes and UFC1 (HPN) (see Supplementary Fig. 7d for protein loading controls). f Western blot showing the effect of UFC1 (HPN) on in vitro ufmylation. UFC1 (HPN) in the presence of DDRGK1-UFL1 did not generate ufmylated product (~40 kDa) (See Supplementary Fig. 7e for protein loading controls). g Western blot showing the effect of UFC1 (HPN) on the ufmylation of UFC1 C116A in trans. See Supplementary Fig. 7f for the protein loading controls. Data in e, f are representative of two independent experiments. Data in g is performed once. Source data are provided as a Source Data file. h Superposition of UBC9 with UFC1 (HPN). For simplicity, only the HPN motif of UBC9 is shown (yellow), along with the helix corresponding to the long helix in UFC1 (blue). While Pro and Asn in the HPN motif of UFC1 (HPN) adopt similar positions as in the UBC9 HPN motif, the His in UFC1 (HPN) adopts a different orientation compared to the His in the UBC9 HPN motif. This difference is likely due to the longer helix in UFC1 that prevents His from reaching the right position.