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J. Biol. Chem., Vol. 280, Issue 29, 27356-27365, July 22, 2005

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

Jana Narasimhan¶, Ming Wang||, Zhuji Fu, Jennifer M. Klein**, Arthur L. Haas**, and Jung-Ja P. Kim

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and the ^**Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112

The biological effects of the ISG15 protein arise in part from its conjugation to cellular targets as a primary response to interferon-/ 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-Å resolution. The ISG15 structure comprises two -grasp folds having main chain root mean square deviation (r.m.s.d.) values from ubiquitin of 1.7 Å (N-terminal) and 1.0 Å (C-terminal). The -grasp domains pack across two conserved 3₁₀ helices to bury 627 Ų 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 Å) 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.

Received for publication, March 15, 2005 , and in revised form, May 19, 2005.

The atomic coordinates and structure factors (code 1Z2M) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by U. S. Public Health Service Grants GM15977 (to J. N.), GM47426 (to A. L. H.), and GM29076 (to J.-J. P. K). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Fig. S1.

Both authors contributed equally to this work.

^¶ Present address: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202.

^|| Present address: W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.

To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-3004; Fax: 504-568-3370; E-mail: ahaas{at}lsuhsc.edu.

To whom correspondence may be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. Tel.: 414-456-8479; Fax: 414-456-6510; E-mail: jjkim{at}mcw.edu.

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