The Hinge Region between Two Ubiquitin-like Domains Destabilizes Recombinant ISG15 in Solution

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• 作者：Christina M. Sorensen ; Lea A. Rempel ; Shane R. Nelson ; Brian R. Francis ; David J. Perry ; Randolph V. Lewis ; Arthur L. Haas ; Thomas R. Hansen
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：January 23, 2007
• 年：2007
• 卷：46
• 期：3
• 页码：772 - 780
• 全文大小：540K
• 年卷期：v.46,no.3(January 23, 2007)
• ISSN：1520-4995

文摘

Interferon-stimulated gene (ISG) 15 mediates antiviral responses and also is upregulated withinthe endometrium in response to the developing embryo during early pregnancy. Structurally, ISG15resembles two ubiquitin domains (30% identical) that are separated by a hinge region. Recombinant (r)bovISG15 is not stable in solution. It was hypothesized that the hinge region contributed to the instabilityof rbovISG15. Within 24 h of dialysis, rbovISG15 formed complexes as detected by reducing and denaturingSDS-PAGE. However, chemical perturbations of cysteine prevented formation of rbovISG15 complexesover time. Furthermore, a site-directed mutant of rbovISG15 (Cys80Ser) was isomeric and more stablethan rbovISG15. Neither wild-type nor mutant rbovISG15 was able to interact with the ISG15 E1 initiatingenzyme, UBE1L, in an in vitro pull-down assay. Ovine (ov) ISG15 has three additional amino acidswithin the hinge region that were hypothesized to increase stability and the degree of interaction withUBE1L because of increased separation of the ubiquitin-like domains. Over time in solution, rovISG15the level of rovISG15 secondary structure was diminished, whereas the Cys80Ser rovISG15 structure didnot change. A GST-Cys80Ser rovISG15 fusion protein had increased structural stability and enhancedprotein-protein interaction with UBE1L after dialysis for 48 h, when compared to the GST-rovISG15fusion protein or rbovISG15. Models of bovISG15, Cys80Ser bovISG15, and ovISG15 were constructed,which confirmed that the hinge region between the two ubiquitin domains destabilizes rbovISG15 insolution.