Fig. 2: The hydrogen bond that T106 forms with the backbone amide of K108 is essential for UFC1 activity.

a UFC1 T106 forms two hydrogen bonds, one with the backbone amide of K108 and one with the side chain of E149. b Super-positioning the crystal structures of UFC1 E149D (yellow) onto WT UFC1 (blue). The loop harboring T106 (residues 103–112) adopts different conformation in the mutant. c The crystal structure of UFC1 E149D shows that the distance between the side chains of T106 and D149 is 7.0 Å, which is too far for hydrogen bonding (indicated by a red dotted line). The N-zeta atom of K108 forms hydrogen bond with the side chain of D149. K108 retains the hydrogen bond with T106, as in the WT. d A representative western blot showing the effect of UFC1 E149I and E149D mutants on in vitro ufmylation (see Supplementary Fig. 3a for protein loading control). e The band corresponding to the ufmylated product (~40 kDa) was quantified for each mutant and normalized against the product obtained in WT protein (data represented as mean ± SD, n = 5 independent experiments). The difference is significant for UFC1 E149D (two‐tailed unpaired Student’s t‐test, p < 0.01) but not for UFC1 E149I. f A representative western blot showing the effect of UFC1 E149I and E149D mutants on RPL26 ufmylation (see Supplementary Fig. 3b for protein loading control). g The band corresponding to the ufmylated RPL26 (RPL26-UFM1) was quantified for each mutant and normalized against RPL26-UFM1 obtained with WT protein (data represented as mean ± SD, n = 3 independent experiments). The difference is significant for UFC1 E149D (two‐tailed unpaired Student’s t‐test, p < 0.05) but not for UFC1 E149I. h Crystal structure for UFC1 T106S. The T106S mutant forms a hydrogen bond with the backbone N of K108. i A representative western blot showing the effect of the UFC1 T106S and T106C mutants on in vitro ufmylation (see Supplementary Fig. 3d for protein loading control). j The band corresponding to the ufmylated product (~40 kDa) was quantified for each mutant and normalized against the product formed in WT protein (data represented as mean ± SD, n = 5 independent experiments). The difference is significant for the T106C (p < 0.0001) but not for the T106S mutant in two‐tailed unpaired Student’s t‐test. k Western blot showing  the  effect of UFC1 T106S and T106C on RPL26 ufmylation (see Supplementary Fig. 3e for protein loading control). Data is representative of two independent experiments. Source data are provided as a Source Data file.