Structural architecture of Galdieria sulphuraria DCN1L.

NEDD8ylation is a major regulatory event for cullin-RING E3 ubiquitin ligases (CRL).1,2 NEDD8ylation activates the ubiquitylation activity of CRLs,3–5 which serves to regulate a wide variety of cellular processes6 by selecting specific targets for destruction in the proteasome.7 NEDD8 E3 ligases catalyze ligation of the ubiquitin-like protein NEDD8 (Rub1 in yeast) to the C-terminal winged-helix domain of the CRL “cullin” subunit.8 NEDD8ylation increases the ubiquitin (Ub) ligase activity of CRLs by enhancing binding of the CRL RING subunit to Ub-charged Ub-conjugating (E2) enzymes,9 and by driving conformational changes that permit the RING subunit to productively orient Ub-E2 conjugates with bound CRL substrates for catalysis.10 However, it is unclear how the NEDD8 E3 ligase catalyses its reaction, what its exact composition is, and how it is regulated. Moreover, current structural and functional models of the NEDD8 E3 ligase are based on the Saccharomyces cerevisiae system,8 in which, an integral NEDD8 E3 ligase subunit, DCN1, has low sequence identity to mammalian DCN1-like proteins, and several amino acid insertions, which together may impede understanding of NEDD8 E3 function in a mammalian system. Protein NEDD8ylation is analogous to ubiquitylation.11 NEDD8-activating enzymes (E1), conjugating enzymes (E2), and ligases (E3) are required for the ligation of NEDD8 to its target CRL(s).2 Each enzyme catalyzes transfer of NEDD8 to its respective acceptor protein through NEDD8’s C-terminal glycine carboxylate. First, in an ATP dependent reaction, NEDD8 is covalently linked to the active site cysteine of a heterodimeric E1 enzyme.12 NEDD8 is then transferred to the active site cysteine of an E2 enzyme (Ubc12).13 The E3 enzyme complex then serves to ensure reaction specificity and to promote chemical catalysis en route to NEDD8 transfer to a specific cullin lysine.8 The NEDD8 enzyme complex contains the cullin substrate, a RING domain containing subunit with bound NEDD8-E2 conjugate, and a DCN1-like protein (DCN1L).8 Mammalian cells utilize five DCN1L paralogs,14 in contrast to only two cullin-associated RING paralogs.8 The exact function of each DCN1L is currently unknown. Some DCN1Ls may work in a non-redundant manner as exemplified by human DCN1L3, which preferably interacts with human cullin 3 (Cul3), and localizes Cul3 to the plasma membrane.14 DCN1Ls generally contain two domains, an N-terminal domain that is unique for each paralog, and a conserved C-terminal domain, termed the PONY-domain.15,16 The purpose of the N-terminal domain is not well understood, but likely aids in reaction specificity and cellular localization.14 The PONY-domain, however, is integral to protein NEDD8ylation, and is functional in CRL neddylation in the absence of the N-terminal domain.8,16 Recently, Scott et al. solved the structure of yeast DCN1 in complex with the winged-helix domain of the S. cerevisiae cullin, Cdc53, and provided some clarification for the composition and mechanism of NEDD8 E3s.8 They identified DCN1 and the S. cerevisiae CRL RING subunit, Hrt1, as the major functional components of the yeast NEDD8 E3 ligase. Cdc53 appears to serve as the scaffold for its own NEDD8ylation, as it is already binds Hrt1 in the canonical CRL complex and binds tightly to DCN1.8 Hrt1 binds the NEDD8-charged E2, Ubc12, and enhances chemical catalysis.8 DCN1 facilitates catalysis, presumably by optimizing the orientation of the NEDD8-Ubc12 conjugate with the reactive Cdc53 lysine residue.8 Here, we report the structure of a DCN1L from Galdieria sulphuraria (Gs-DCN1L) to 1.3 A resolution. As all DCN1L structures deposited in the PDB to date are from S. cereviseae DCN1 (Sc-DCN1), which has low sequence identity and frequent amino acid insertions within its polypeptide chain as compared to mammalian DCN1Ls, we sought a DCN1L that would be more comparable with mammalian forms. Gs-DCN1L was identified in genomic sequences17 and expressed sequence tag data from G. sulphuraria.18 Gs-DCN1L was targeted for structural characterization when crystallization trials for the homologous protein, Mus musculus Dcn1D1 (Mm-Dcn1D1), failed to yield crystals. Gs-DCN1L shares higher sequence identity with mammalian DCN1Ls than Sc-DCN1, with a low frequency of amino acid insertions and gaps. Gs-DCN1L shares 25% amino acid identity with Sc-DCN1. At the cullin-binding region Gs-DCN1L and Sc-DCN1 are very similar in fold and in sequence, indicative of a conserved cullin-binding mode. Gs-DCN1L and Sc-DCN1 structures diverge markedly near the cullin interaction surface. In this region Gs-DCN1L has a disordered loop that exposes a hydrophobic pocket, whereas, the comparable polypeptide segment of Sc-DCN1 completely covers the analogous hydrophobic pocket, and is well ordered.8,15,16 Sequence analysis indicates that this intriguing structural divergence is conserved among Gs-DCN1L and the mammalian DCN1L paralogs and may provide clues for additional DCN1L functionalities.