Fig. 4: Biochemical characterization of ISG15 specificity.
From: Insights into the ISG15 transfer cascade by the UBE1L activating enzyme
a Structure of ISG15 (pdb 1z2m) highlighting residues that contact UBE1L. Residues are shown in ball-and-stick representation under a semi-transparent surface. Interactions are exclusively located in the C-terminal ubiquitin-like fold (ISG15 C-lobe; also see Fig. 3). Analogous residues of ubiquitin are shown in teal. b Diagram representing ISG15 constructs used in c–g. From left to right: ISG15C-lobe, full-length ISG15 (ISG15FL), ubiquitin(Ub)-ISG15C-lobe fusion, SUMO1-ISG15C-lobe fusion, ISG15 mutants (ISG15mut(s)). c UBE1L charging reactions with fluorescent ISG15 and ISG15C-lobe. Reactions were separated by SDS-PAGE and visualized with fluorescent imaging. d UBE1L charging reactions with ISG15FL, Ub-ISG15C-lobe, and SUMO1-ISG15C-lobe. Reactions were separated by SDS-PAGE and visualized with Coomassie stain. e Quantification of UBE1L charging reactions with fluorescent ISG15 and ISG15 mutants. ISG15 mutants include: ISG153xmut (T125I/ F149H/N151V), ISG154xmut (T125I/P130Q/F149H/N151V), ISG155xmut (W123R/T125I/P130Q/F149H/N151V). Mutated ISG15 residues were swapped with the analogous residues of ubiquitin (also see Supplementary Fig. 5g). Reactions were performed as in c. Error values represent s.d. from the mean (n = 3 independent experiments). Samples derive from same experiment and gels were processed in parallel. f Quantification of UBA1 charging with ISG15 mutants as in e. Error values represent s.d. from the mean (n = 3 independent experiments). Samples derive from same experiment and gels were processed in parallel. g UBE1L and UBA1 charging reactions with fluorescent ISG15FL and ISG155xmut as in c. Experiments were performed independently in triplicate. Source data are provided in the Source Data file.
