Fig. 3: C-alpha hydrogen bonding plays an important role in oxyanion stabilization.

a B-factor analysis of the K108 C-alpha in UFC1 WT and mutants. b A representative western blot showing the effect of the UFC1 T106I/W145H double mutant on in vitro ufmylation, in comparison to the T106I or W145H mutants (see Supplementary Fig. 4h for protein loading control). c The band corresponding to the ufmylated product (~40 kDa) was quantified for each mutant and normalized to the product formed in WT protein (data represented as mean ± SD, n = 4 independent experiments). The difference was significant for the T106I/W145H double mutant (p < 0.0001, two-tailed unpaired Student’s t-test) when compared with the T106I mutant. The difference was significant for the W145H mutant when compared with WT (p < 0.01, two-tailed unpaired Student’s t-test). d Super-position of UBCH5B charged with Ub (PDB 3a33) onto the structure of UFC1. UFC1 active site C116 is super-imposed onto S95 of UBCH5B (the presence of a serine instead of a cysteine at this position allows the formation of an oxy-ester bond with Ub (white)). The C-terminal carboxyl of Ub faces the C-alpha of UFC1 K108, allowing C-alpha hydrogen bonding. e Mimicry of the oxyanion intermediate through a Cys to Glu replacement. The side chain of Glu places a negatively charged oxygen at a distance from the Glu C-alpha that resembles the distance between the C-alpha of the active site Cys and the negatively charged oxygen of the Ub/UBL carbonyl during the nucleophilic attack. f Crystal structure of UFC1 C116E showing the hydrogen bond between the Glu side chain and the C-alpha of K108; and the distance between the Glu side and the backbone amide of M109. g B factor analysis of M109 C-alpha in the indicated mutants. The presence of E116 reduces the B-factor of M109 C-alpha. h Crystal structure of UFC1 T106I/C116E showing the distance between K108 C-alpha and E116 side chain oxygen. i B-factor analysis of the K108 C-alpha in the indicated UFC1 mutants.