br Results br Discussion Taking advantage
Discussion Taking advantage of the highly specific protein–protein interactions among cognate Dequalinium Chloride that mediate SUMO conjugation to substrates, we have developed a novel strategy for achieving inhibition of SUMO conjugation in vivo based on disruption of SUMO E1–E2 interactions. We have validated this strategy for uncovering a novel role of SUMO conjugation in defense responses to necrotrophic fungal pathogens.
Funding This work was supported by the European Research Council (ERC-2007-StG-205927) and the Spanish Ministry of Science (BIO2008-01495). L.C.-M., I.T., A.P., S.M., and N.R. were supported by research contracts through the CRAG. A.M. and J.S. were supported by predoctoral fellowships, Spanish Ministry of Education, Culture and Sport (FPU12/05292) and Ministry of Education and Science (BES-2005-6843), respectively, and A.L.S. was supported by Beatriu de Pinós post-doctoral grant of the Generalitat de Catalunya (2013 BP_B 00182). We also thank the Generalitat de Catalunya (Xarxa de Referència en Biotecnologia and 2009SGR 09626) for substantial support.
Introduction The covalent attachment of the small, highly conserved protein ubiquitin (Ub) to target proteins is an essential mechanism for modulating the function of these proteins in eukaryotes, and its defective regulation has been shown to cause pathological conditions that range from developmental abnormalities and cancer to autoimmune and neurodegenerative diseases (Hershko and Ciechanover, 1998). Ub is covalently conjugated via its C terminus to its cellular target proteins by a conserved biochemical pathway that involves the sequential actions of Ub-activating (E1), Ub-conjugating (E2), and Ub-protein ligase (E3) enzymes (Hershko and Ciechanover, 1998). The E1 activity represents an essential step during Ub and ubiquitin-like protein (Ubl) conjugation, and the general mechanism of the E1-catalyzed reaction is well established, on the basis of early work with the Ub-E1 (Haas and Rose, 1982, Haas et al., 1982, Hershko et al., 1983). Each Ubl has a dedicated E1 that initiates its conjugation cascade. First, E1 associates with the Ubl and catalyzes the adenylation of the Ubl C terminus in an ATP-dependent process. Second, E1 forms a thioester between a conserved catalytic cysteine and the Ubl. Next, E1 is loaded with a second Ubl molecule, followed by its C-terminal adenylation. Finally, the ternary E1∼Ubl thioester complex recruits an E2 to facilitate transfer of the thioester-linked Ubl to a conserved E2 cysteine (transthioesterification). The energy stored in the E2∼Ubl thioester is utilized to conjugate Ubl to target lysine ɛ-amino groups, either directly or through complexes mediated by E3s. The E1 for Ub is a 110–120 kDa monomeric protein, whereas the E1s for the Ubls NEDD8 and SUMO are heterodimeric complexes with comparable overall molecular weights. All eukaryotic E1s contain a two-fold repeat of a domain that is derived from the bacterial MoeB and ThiF proteins (Johnson et al., 1997), with one occurrence each in the N-terminal and C-terminal half of the E1 for Ub, or the separate subunits of the E1 for NEDD8 and SUMO. MoeB and ThiF catalyze the C-terminal adenylation of MoaD and ThiS, respectively, which are structural homologs of Ubl in the context of either molybdenum cofactor or thiamin biosynthesis (Duda et al., 2005, Lake et al., 2001, Lehmann et al., 2006). The Ub-E1 consists of four building blocks: First, the adenylation domains composed of two MoeB/ThiF-homology motifs (hereafter, IAD and AAD, for “inactive” and “active” adenylation domain, respectively), the latter of which binds ATP and Ub (Lake et al., 2001, Lois and Lima, 2005, Walden et al., 2003b); second, the catalytic cysteine half-domains, which contain the E1 active site cysteine (hereafter, FCCH and SCCH, for “first” and “second” catalytic cysteine half-domain, respectively) inserted into each of the adenylation domains (Szczepanowski et al., 2005); third, a four-helix bundle that represents a second insertion in the IAD and immediately follows the FCCH; and fourth, the C-terminal ubiquitin-fold domain (UFD), which recruits specific E2s (Huang et al., 2005, Huang et al., 2007, Lois and Lima, 2005). Despite the central role of the E1 enzyme in the Ub conjugation cascade, a structure of an intact Ub-activating enzyme has not been available so far. Here, we present the crystal structure of the Saccharomyces cerevisiae Ub-activating enzyme, known as Uba1, bound to yeast Ub, and on the basis of structural and biochemical data advance our understanding of the function and mechanism of the Ub-specific E1.