Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies

Swatek, Kirby N.; Aumayr, Martina; Pruneda, Jonathan N.; Visser, Linda J.; Berryman, Stephen; Kueck, Anja F.; Geurink, Paul P.; Ovaa, Huib; van Kuppeveld, Frank J. M.; Tuthill, Tobias J.; Skern, Tim; Komander, David

Structure of Lb pro Bound to ISG15. To decipher the mechanism of this unprecedented activity, we designed an ISG15 suicide probe specific for Lb pro . As mentioned, the WT ISG15 suicide probe modified several viral proteases but was too long to modify Lb pro (Fig. S2E ). Removing the GlyGly motif from ISG15 CTD and replacing Arg155 with a Gly-like C-terminal propargyl warhead ( Fig. 2A ) generated an ISG15 CTD -ΔC probe that efficiently modified Lb pro (Fig. 2B and Fig. S2E ). This enabled purification, crystallization, and structure determination of a covalent Lb pro ∼ISG15 CTD -ΔC complex at 1.5-Å resolution (Fig. 2 C and D, Fig. S3 , and Table S1 ). As anticipated, the ISG15 CTD -ΔC probe had covalently modified the catalytic Cys51 of Lb pro (Fig. S3) . Individually, the Lb pro protease and ISG15 domains were similar to previously determined structures (18, 23) (Cα rmsds of 0.609 and 0.274 Å, respectively). The structure revealed how proteolytic cleavage of ISG15 is achieved ( Fig. 2E and Fig. S3 ). Key interactions are formed via the ISG15 C terminus and also, via a hydrophobic surface centered on ISG15 Trp123 (Fig. S3) . The positively charged residues of the ISG15 C terminus, Arg153 and Arg155 (the latter mutated to Gly in the ISG15 CTD -ΔC probe), are cradled by an acidic groove that consists of Asp49, Glu96, and Glu147 on Lb pro (Fig. S3G) . Furthermore, ISG15 Leu154 occupies a hydrophobic pocket commonly found in papain-like enzymes. Mutation of either Leu154 or Arg155 to Ala in proISG15 strongly reduced cleavage by Lb pro (Fig. S3H) . However, these C-terminal mutations do not prevent ISG15 binding, since the ISG15 CTD -ΔC suicide probe that lacks Arg155 was able to modify Lb pro (Fig. 2 B and C) . The importance of the C terminus explains, at least in part, the observed weak cross-reactivity with ubiquitin and NEDD8, which feature identical or similar C-terminal sequences, respectively, and also, the inability of Lb pro to target other modifiers, such as SUMO1, which feature more divergent C-terminal sequences (24) (Fig. S3F) . The observed interactions closely mimic those seen in the previously determined structure of Lb pro carrying out self-processing of its own C terminus (23) (Fig. 2E and Fig. S3F ).