Crystal Structure of the Interferon-induced Ubiquitin-like Protein ISG15

Abstract

The biological effects of the ISG15 protein arise in part from its conjugation to cellular targets as a primary response to interferon-alpha/beta induction and other markers of viral or parasitic infection. Recombinant full-length ISG15 has been produced for the first time in high yield by mutating Cys78 to stabilize the protein and by cloning in a C-terminal arginine cap to protect the C terminus against proteolytic inactivation. The cap is subsequently removed with carboxypeptidase B to yield mature biologically active ISG15 capable of stoichiometric ATP-dependent thiolester formation with its human UbE1L activating enzyme. The three-dimensional structure of recombinant ISG15C78S was determined at 2.4-A resolution. The ISG15 structure comprises two beta-grasp folds having main chain root mean square deviation (r.m.s.d.) values from ubiquitin of 1.7 A (N-terminal) and 1.0 A (C-terminal). The beta-grasp domains pack across two conserved 3(10) helices to bury 627 A2 that accounts for 7% of the total solvent-accessible surface area. The distribution of ISG15 surface charge forms a ridge of negative charge extending nearly the full-length of the molecule. Additionally, the N-terminal domain contains an apolar region comprising almost half its solvent accessible surface. The C-terminal domain of ISG15 was superimposed on the structure of Nedd8 (r.m.s.d. = 0.84 A) bound to its AppBp1-Uba3 activating enzyme to model ISG15 binding to UbE1L. The docking model predicts several key side-chain interactions that presumably define the specificity between the ubiquitin and ISG15 ligation pathways to maintain functional integrity of their signaling.

Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp

“…One is lack of the Cys residues in the linker sequences of fish proteins, which is conserved in mammalian proteins and is essential for the stabilization of ISG15 [6]; the other is that fish proteins, together with ruminant ISG15 proteins, have not any additional C-terminal amino acids following the highly conserved ISG15 C-terminal sequence ''LRLRGG'' [2,23]. Like ubiquitin, ISG15 can be conjugated to target proteins via its C-terminal end [7].…”

“…For the induction by Poly I:C (Sigma) or LPS (Sigma), cells were directly incubated with 5 ml of FCS-free medium containing 100 mg ml À1 of Poly I:C or 50 mg ml À1 of LPS, respectively. CAB cells were harvested at 1, 3,6,8,12,24,48, and 72 h post treatment, and total RNA was extracted with RNA-Solv Reagent (Omega Biotek). Contaminating genomic DNA in the RNA samples was thoroughly digested by DNase without RNase (Promega) before RNA was treated by chloroform and subsequently precipitated by ethanol.…”

“…Within the 3 0 untranslated regions, two AU sequences (ATTTA), a motif involved in rapid message degradation [29], were found in CaISG15-1 cDNA and six in CaISG15-2 cDNA. The putative CaISG15-1 and CaISG15-2 proteins have two tandem ubiquitin-like domains, and each domain has six invariant aliphatic residues that were crucial for the hydrophobic core of the b-grasp fold of ubiquitin [6]. No LRLRGG motif is found in the end of the first ubiquitin-like domain, which is distinct from polyubiquitin.…”

“…Most of these proteins show significant sequence homologous to ubiquitin and contain single ubiquitin-like (UBL) domains, while ISG15 is composed of two in tandem, each of which bears approximately 30% homology to ubiquitin [5]. A recent solved crystal structural analysis revealed that both ubiquitin-like folds of human ISG15 are joined by a six-residue extended linker sequence [6]. Similar to ubiquitin, ISG15 contains the canonical LRLRGG motif at its C terminus [2].…”

Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6

“…This cyclized FAT10 species is no longer a substrate for Uba6, which complicates the stoichiometric and kinetic analysis of its activation by Uba6 (supplemental Table 1). Therefore, the FAT10 mutant was constructed for this study following a strategy adopted for studies of ISG15, a UBL that contains two ubiquitin-like domains with an internal reactive cysteine (29).…”