Figure 3. Role of the Lys48 face in ubiquitin chain synthesis.

(a) Schematic showing the Lys48 face of ubiquitin with residues predicted to interact with the active site of Ube2K indicated in black. Beige-coloured text indicates residues on the surface that are not predicted to be involved in binding to Ube2K. Boxed residues have been mutated in this study. Carbon, nitrogen and oxygen atoms are coloured beige, blue and red, respectively. (b) Sequence of ubiquitin showing the elements of secondary structure, with mutated residues indicated (+). (c) Single turnover diUb assay (40 minutes) where the ubiquitin variant is either Ub^A (top) or Ub^D (bottom). The presence of diUb indicates the ubiquitin variant can support chain formation. Reducing and non-reducing samples are shown to evaluate charging.