Figure 6. Residues 85–88 are critical for diUb synthesis.

(a) Ube2K protein variants containing mutations targeting residue 85 were used in 40 minute diUb (upper; reducing gels) and charging reactions (lower; non-reducing gels). (b–d) DiUb and charging reactions as in panel A with Ube2K protein variants containing mutations to residues 86 (b), 87 (c), and 88 (d). (e) Schematic of the molecular model with a close up view of the predicted interaction between Ub^A and Ube2K~Ub. Interacting side-chains are shown as sticks and hydrogen bonds are indicated with dashed lines. Oxygen, nitrogen and sulphur atoms are red, blue and yellow, respectively. Carbon atoms are coloured as in the schematic above.