Extended Data Figure 2. Engineered Lb^pro has enhanced activity against ubiquitin.

a, Left, structural model of ubiquitin-bound Lb^pro as in Extended Data Fig. 1c with ubiquitin under a green surface. Top right, close-up view of the ubiquitin Ile44 patch (Leu8, Ile44, His68, Val70) and its predicted interactions with Lb^pro. Differences in the equivalent surface in ISG15 explain its higher affinity ¹⁹. Bottom right, modeling of an improved hydrophobic contact between Lb^pro and ubiquitin. The corresponding L102W Lb^pro mutant is denoted as Lb^pro*. b, Diubiquitin cleavage assays, as in Fig. 1a. The cleavage of each diubiquitin (diUb) linkage type was compared for Lb^proand Lb^pro*. Assays were performed in duplicate. c, Example ubiquitin-KG-TAMRA cleavage assays comparing Lb^pro (left) and Lb^pro*(right), the difference in polarization relative to a substrate-only negative control is shown. The average trace of assays performed in technical triplicate is shown. TAMRA-KG represents a cleaved product positive control. d, Catalytic efficiencies derived from two independent sets of ubiquitin-KG-TAMRA cleavage measurements as in c. Slope and errors values derived from linear regression are reported for each replicate individually. Fold improvement of Lb^pro* over Lb^pro in catalytic efficiency towards this substrate is indicated. e, Example diubiquitin K63-FlAsH cleavage assays as in c. f, Catalytic efficiencies derived from two independent sets of diubiquitin K63-FlAsH cleavage measurements as in e. Slope and errors values derived from linear regression are reported for each replicate individually. Fold improvement of Lb^pro* over Lb^pro in catalytic efficiency towards this substrate is indicated. The REP1 [Lb^pro] = 7.81μM data point has been excluded, as reliable exponential decay parameters could not be fitted for this point. g, Branched ubiquitin cleavage assays. Three different branched ubiquitin chains (Lys6/Lys48; Lys11/Lys48; Lys11/Lys63, described in ⁴⁹) were used in Lb^proand Lb^pro* cleavage assays. Assays were performed in triplicate. h, Catalytic efficiencies derived from gel-based analysis of three independent Lys6/Lys48 branched triubiquitin cleavage assays performed at three different enzyme concentrations. Fold improvement of Lb^pro* over Lb^pro in catalytic efficiency towards this substrate is indicated. Centred values correspond to the mean of independent experiments performed in triplicate. Error values represent s.d. from the mean. Slope and errors values derived from linear regression based on the mean values are reported. For gel source data, see Supplementary Fig. 1.