Structural basis of the specificity of USP18 toward ISG15

Abstract

Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin- and Ubl-proteases collectively called DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity towards ubiquitin but specifically deconjugates the interferon induced Ubl ISG15. To identify molecular determinants for this specificity, we solved the crystal structures of mouse USP18 and of mouse USP18 in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15 providing evidence that ISG15 specificity of USP18 is mediated by a small interaction interface. Our results may provide the structural basis for the development of new drugs modulating ISGylation.

Extracellular ISG15 Signals Cytokine Secretion through the LFA-1 Integrin Receptor

“…Figure 2C shows that the C-lobe residues identified in this analysis cluster on the surface of ISG15, comprising what we will refer to as the “IFN-γ patch” (see also Supplemental Figure S1A). The IFN-γ patch does not correspond to a region that mediates interaction with other known ISG15 binding proteins, including influenza virus NS1B (Guan et al, 2011) or USP18 (Basters et al, 2017), and does not correspond to the “hydrophobic patch” region of ubiquitin, which is the binding site for many ubiquitin binding proteins (Sloper-Mould et al, 2001) (Supplemental Figure S1B, C). …”

Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease

“…In ISG15, however, this hydrophobic 'patch' appears to be extended by Trp123 and Pro130, thus allowing His1652 of PLpro to also engage in an interaction. As previously noted by Basters et al for USP18, this region of ISG15 is conserved across different ISG15 species and was proposed to be a unique hydrophobic patch to ISG15 differing from the well-known hydrophobic patch of Ub, which is commonly utilized for recognition by a vast number of Ub-binding proteins (Basters et al, 2017;Komander and Rape, 2012). Our observations and those of others collectively support the hypothesis that this hydrophobic region of ISG15 may be a canonical recognition region that is utilized in general by deISGylases to specifically recognize ISG15.…”

“…Trp123 of ISG15 is sandwiched between the aliphatic chain of Arg1649 of PLpro and the hydrophobic side-chain of Val1691, which also interacts with the alkyl group of Thr125 of ISG15. Apart from Thr125, which is unique to human ISG15, the proline and tryptophan residues are conserved among species of ISG15, and appear to be utilized for interactions by a wide range of deISGylases, both in the USP and ovarian tumor family (vOTU), suggesting that this region of PLpro may be critical for ISG15 recognition (Akutsu et al, 2011;Basters et al, 2017;Daczkowski et al, 2017a).…”

“…The X-ray structure of mouse USP18 in complex with full-length mouse ISG15 has been determined, but USP18 has low sequence identity to PLpros. In addition, USP18 strictly recognizes ISG15 and has no recognition of Ub (Basters et al, 2017).…”

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

“…Daczkowski et al (2017a) reported that the C-terminal domains of ISG15s (similar to Ub1 mentioned above) from different species have different binding characteristics with SARS-CoV PL2 pro according to two structures, the PL2 pro in complex with the C-terminal domain of hISG15 and mISG15, respectively. In addition, the structure of mouse USP18 in complex with full-length mISG15 became available this year (Basters et al, 2017). Surprisingly, the N-terminal Ubl domain of mISG15 shows almost no interaction with mUSP18.…”