title: 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.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Jana
authors.initials: J
authors.lastname: Narasimhan
authors.forename: Ming
authors.initials: M
authors.lastname: Wang
authors.forename: Zhuji
authors.initials: Z
authors.lastname: Fu
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Klein
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Haas
authors.forename: Jung-Ja P
authors.initials: JJ
authors.lastname: Kim
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D016207:Cytokines / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins; D055672:Static Electricity; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism
chemical_list: D016207:Cytokines; D011994:Recombinant Proteins; C471538:UBA1 protein, human; D014452:Ubiquitins; C074034:ISG15 protein, human; D044764:Ubiquitin-Activating Enzymes