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Structural Basis for the Removal of Ubiquitin and Interferon-Stimulated Gene 15 by a Viral Ovarian Tumor Domain-Containing Protease

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Structural Basis for the Removal of Ubiquitin and Interferon-Stimulated Gene 15 by a Viral Ovarian Tumor Domain-Containing Protease Structural basis for the removal of ubiquitin and interferon-stimulated gene 15 by a viral ovarian tumor domain-containing protease Terrence W. Jamesa,1, Natalia Frias-Stahelib,1,3, John-Paul Bacika, Jesica M. Levingston Macleodb, Mazdak Khajehpourc, Adolfo García-Sastreb,d,e, and Brian L. Marka,2 aDepartment of Microbiology, and cDepartment of Chemistry, University of Manitoba, Winnipeg, MB, R3T 2N2 Canada; and bDepartment of Microbiology, dDepartment of Medicine, Division of Infectious Diseases, and eGlobal Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, NY 10029 Edited by J. Wade Harper, Harvard, Boston, MA, and accepted by the Editorial Board December 7, 2010 (received for review September 7, 2010) The attachment of ubiquitin (Ub) and the Ub-like (Ubl) molecule of cellular target proteins similarly to Ub (9). Although conjuga- interferon-stimulated gene 15 (ISG15) to cellular proteins mediates tion is essential (8), several possible antiviral mechanisms have important innate antiviral responses. Ovarian tumor (OTU) domain recently been proposed for ISG15 (10). proteases from nairoviruses and arteriviruses were recently found Ub and ISG15 conjugation can be reversed by deubiquitinating to remove these molecules from host proteins, which inhibits Ub enzymes (DUBs). Ovarian tumor (OTU) domain proteases are and ISG15-dependent antiviral pathways. This contrasts with the papain-like cysteine DUBs that have been identified in eukar- Ub-specific activity of known eukaryotic OTU-domain proteases. yotes, bacteria, and viruses (11). We have assayed a number of Here we describe crystal structures of a viral OTU domain from eukaryotic OTU-domain-containing proteins for deubiquitinat- the highly pathogenic Crimean–Congo haemorrhagic fever virus ing and deISGylating activity, including human A20, Cezanne, (CCHFV) bound to Ub and to ISG15 at 2.5-Å and 2.3-Å resolution, otubain1, and otubain2 (12) and the Saccharomyces cerevisiae respectively. The complexes provide a unique structural example of OTU-domain-containing protein Otu1 (see Results and Discus- sion ISG15 bound to another protein and reveal the molecular mechan- ). While these eukaryotic proteins exhibit deubiquitinating BIOCHEMISTRY ism of an ISG15 cross-reactive deubiquitinase. To accommodate activity as expected, none have been found to exhibit deISGylat- structural differences between Ub and ISG15, the viral protease ing activity, suggesting that eukaryotic OTU domains are Ub- binds the β-grasp folds of Ub and C-terminal Ub-like domain of specific. Importantly, the deubiquitinating activities of human ISG15 in an orientation that is rotated nearly 75° with respect to A20, and the OTU-domain-containing protein DUBA, have been that observed for Ub bound to a representative eukaryotic OTU found to down-regulate TNFα and type I IFN production, respec- domain from yeast. Distinct structural determinants necessary tively, by removing Ub from signaling proteins (13, 14). for binding either substrate were identified and allowed the reen- In contrast to the Ub-specific activity of known eukaryotic gineering of the viral OTU protease into enzymes with increased OTU domains, we recently found that OTU-domain-containing substrate specificity, either for Ub or for ISG15. Our findings now proteins from nairoviruses and arteriviruses, two unrelated provide the basis to determine in vivo the relative contributions of groups of RNA viruses, act as ISG15 cross-reactive deubiquiti- deubiquitination and deISGylation to viral immune evasion tactics, nases, removing both Ub and ISG15 from host proteins (12). and a structural template of a promiscuous deubiquitinase from a CCHFV is a nairovirus that causes severe haemorrhagic fever haemorrhagic fever virus that can be targeted for inhibition using in humans, with a mortality rate approaching 30% (15). As for small-molecule-based strategies. other members in the genus, its genome consists of three nega- tive-sense RNA segments, the largest of which encodes a 448 kDa innate immunity ∣ viral deubiquitinase ∣ bunyavirus ∣ Crimean–Congo viral RNA polymerase (L) that contains an amino terminal OTU haemorrhagic fever virus ∣ viral immune evasion domain of approximately 18 kDa (16, 17). Arteriviruses, such as equine arteritis virus (EAV) and porcine respiratory and repro- he posttranslational modification of proteins by Ub and ductive syndrome virus (PRRSV), are nonsegmented, positive- TUb-like (Ubl) molecules is a regulatory process that controls sense RNA viruses that contain an OTU domain within their numerous biological events (1, 2). Ub is conjugated to a lysine nonstructural protein 2 (nsp2) (18). The catalytic activity of these residue of target proteins through an isopeptide bond between viral OTU domains not only inhibits NF-κB-dependent signaling the terminal carboxyl group of Ub and ϵ-amino group of the (12, 19), we found that expression of the OTU domain from target lysine (3). Additional Ub molecules can be conjugated CCHFV antagonizes the antiviral effects of ISG15, suggesting to lysines within Ub itself to form polyubiquitin chains. Lys 48-linked polyubiquitination is the canonical signal that targets Author contributions: A.G.-S. and B.L.M. designed research; T.W.J., N.F.-S., J.-P.B., J.M.L.M., proteins for proteasomal degradation, whereas Lys 63-linked and B.L.M. performed research; T.W.J., N.F.-S., J.-P.B., M.K., A.G.-S., and B.L.M. analyzed polyubiquitination can initiate proteasome-independent events data; and T.W.J. and B.L.M. wrote the paper. (4). Both Lys 48- and Lys 63-linked polyubiquitination have been The authors declare no conflict of interest. established as key signaling events that activate innate and adap- This article is a PNAS Direct Submission. J.H. is a guest editor invited by the Editorial Board. tive immune responses (5). Freely available online through the PNAS open access option. A critical innate immune response to viral infection is the Data deposition: The coordinates and structure factors for the CCHFV–OTU–Ub and rapid production of type I interferon (IFN) and tumor necrosis CCHFV–OTU–ISG15 protein complexes have been deposited in the Protein Data Bank, factor alpha (TNFα). The induction and activity of these antiviral www.pdb.org (PDB ID codes 3PT2 and 3PSE, respectively). cytokines is controlled by, among other factors, Ub and Ubl 1T.W.J. and N.F.-S. contributed equally to this work. conjugation (4). A hallmark of type I IFN stimulation is the rapid 2To whom correspondence should be addressed. E-mail: [email protected]. production of the Ubl molecule ISG15 (6). ISG15 is composed 3Present address: Laboratory of Virology and Infectious Disease, The Rockefeller University, of two tandem Ub-like folds (7) and is known to exhibit potent 1230 York Avenue, Box 64, New York, NY 10065. antiviral activity against several important viruses (8). It conju- This article contains supporting information online at www.pnas.org/lookup/suppl/ gates to ϵ-amino groups of target lysine residues on the surface doi:10.1073/pnas.1013388108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1013388108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 28, 2021 that viral OTU domains act as virulence factors to suppress host A B Region1 Leu71 inflammatory and antiviral responses (12). Ubiquitin A number of eukaryotic OTU-domain structures have been Ile131 Glu78 determined (20–23), including a covalent complex between the OTU domain from the Saccharomyces cerevisiae protein Otu1 Region3 Region2 2.9 2.7 Arg72 β Leu73 Thr102 1 2.9 and Ub (24). The structure of a viral OTU domain has not been 3.1 β2 α reported, however, and the mechanism of ISG15 cross-reactivity 5 His146 Ser101 β5 has remained unknown. Here we report crystal structures of the α1 2.8 Arg74 β4 3.1 3.4 viral OTU-domain protease domain from CCHFV covalently α β α6 Gly75 Gly100 2 6 α7 3.2 bound to Ub and to ISG15 at 2.5-Å and 2.3-Å resolution, respec- β7 3CN Glu98 α 3.0 tively. The complexes provide a unique structural example of β3 α3 4 Trp99 ISG15 bound to another protein and together reveal the mole- CCHFV Region1 3CN cular mechanism of an ISG15 cross-reactive deubiquitinase. OTU domain Cys40 The expanded substrate-specificity arises from a unique topolo- Region2 Region3 gical feature of the viral OTU-domain fold, which causes the C Gln49 D protein to bind Ub and C-terminal Ub-like domain of ISG15 in 3.6 Leu71 Ile44 an orientation that is rotated nearly 75° with respect to that ob- Arg42 Leu8 3.0 Pro77 Val70 Leu73 served for Ub bound to a representative eukaryotic OTU domain 3.1 3.3 Arg80 Ile13 Val12 Gln16 Ala129 from yeast. This permits the viral OTU domain to accommodate α Val18 5 Ile131 additional bulk within the C-terminal domain of ISG15 that is not Arg72 Glu78 present in Ub. Individual residues within the substrate-binding 2.7 site of viral protease were identified as key interactions sites for Ub or ISG15, which allowed the reengineering of the protease Fig. 1. Crystal structure of CCHFV–OTU (cyan) bound to Ub (orange). into enzymes with increased substrate specificity, either for Ub or (A) Ribbon diagram of the complex highlighting the three major regions for ISG15. By identifying the unique structural determinants of interaction between Ub and the protease. Ub is bound within a sub- that underlie ISG15 cross-reactivity, our findings now provide the strate-binding groove that is flanked on either side by the β-sheet lobe (left) basis to determine in vivo the relative contributions of deubiqui- and α-helical lobe (right) of the protease. The groove narrows into a channel tination and deISGylation to viral immune evasion tactics and that directs the C terminus of Ub toward the enzyme active site. Gly 76 of Ub the potential to develop small-molecule based strategies that is replaced with a propylamine linker (3CN) that covalently attaches Ub to the α specifically inhibit viral OTU-domain proteases.
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