Abstract
ISG15 (interferon-stimulated gene 15), the first ubiquitin-like protein (UBL) identified, has emerged as an important cellular antiviral factor. It consists of two UBL domains with a short linker between them. The covalent attachment of ISG15 to host and viral proteins to modify their functions, similar to ubiquitylation, is named ISGylation. Influenza B virus NS1B protein antagonizes human but not mouse ISGylation because NS1B exhibits species specificity; it only binds human and non-human primate ISG15. Previous studies have demonstrated that the N-terminal UBL domain and linker of ISG15 are required for the binding by NS1B and that the linker plays a large role in the species specificity, but the structural basis for them has not been elucidated. Here we report the crystal structure of human ISG15 in complex with NS1B at a resolution of 2.0 Å. A loop in the ISG15 N-terminal UBL domain inserts into a pocket in the NS1B dimer, forming a high affinity binding site. The nonspecific van der Waals contacts around the ISG15 linker form a low affinity site for NS1B binding. However, sequence alignment reveals that residues in the high affinity site are highly conserved in primate and non-primate ISG15. We propose that the low affinity binding around the ISG15 linker is important for the initial contact with NS1B and that the stable complex formation is largely contributed by the following high affinity interactions between ISG15 N-terminal UBL domain and NS1B. This provides a structural basis for the species-specific binding of ISG15 by the NS1B protein.
Highlights
Type I interferon production represents one of the first lines of defense against invading viral pathogens [1, 2]
Pathogens induce the expression of hundreds of interferon-stimulated genes (ISGs),2 many of which help establish the antiviral responses in target cells [2, 3]
Influenza B virus strongly induces ISG15 during infection but blocks protein ISGylation by the NS1B protein, which interacts with ISG15 and inhibits its conjugation to target proteins in vitro as well as in infected cells [14]
Summary
Protein Expression and Purification—All constructs expressing the NS1B N-terminal fragment, human ISG15, and its mutants were generated using a standard PCR-based cloning strategy. ISG15 and its mutants with an N-terminal glutathione S-transferase (GST) tag were collected with glutathione-Sepharose 4 Fast Flow (GE Healthcare) followed by digestion with PreScission protease (GE Healthcare) to remove the tag and. REPORT: Complex Structure of Human ISG15 with NS1B further purification by Superdex 200 HR 10/30 gel filtration (GE Healthcare). Gel Filtration Analysis—Size-exclusion chromatography with a Superdex 200 10/300 column on an A KTA FPLC (GE Healthcare) was carried out to access the apparent molecular mass of the ISG15-NS1B complex in solution at 4 °C. For the collection of data for kinetic or equilibrium analyses, ISG15 and all its mutants in a buffer of 0.01 M HEPES, pH 7.2, and 0.5 M NaCl, plus 0.005% (v/v) Tween 20, were injected over the flow cells at various
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