Huang, D.T. et al. Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity. Nature 445, 394-398

Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Nature (Impact Factor: 41.46). 02/2007; 445(7126):394-8. DOI: 10.1038/nature05490
Source: PubMed


Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1 throught UBL thioester intermediate, and generating a thioester-linked E2 throught UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2 throught NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.

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Available from: Danny Huang, Aug 20, 2015
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    • "Mammalian UbE1 is also a large multi-domain enzyme with unknown structure, but the 3D structures of its orthologs have been solved [27], [28]. The domain architecture of human UbE1 shows that it is mainly comprised of the adenylation domain (AD), the catalytic cysteine domains (FCCH, SCCH) and the C-terminal UFD domain (Fig. 5A) [29]. "
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    ABSTRACT: The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.
    PLoS ONE 09/2014; 9(9):e107509. DOI:10.1371/journal.pone.0107509 · 3.23 Impact Factor
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    • "Their E1 enzymes are also homologous to each other in both peptide sequences and crystal structures, except that NAE is a heterodimer of two subunits, APPBP1 and Uba3, while UAE is composed of a single chain [8], [20], [21]. The crystal structures of the E1s bound to their cognate Nedd8 and UB proteins show that Nedd8 and UB are docked to the E1s in a similar mode with their C-terminal peptide extending into the adenylation domains of the E1s to reach to the ATP molecule bound to the E1s [20], [22]. The C-terminal peptides of Nedd8 and UB have the sequences 71LALRGG76 and 71LRLRGG76, respectively, with just one residue at position 72 being different. "
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    ABSTRACT: The C-terminal peptides of ubiquitin (UB) and UB-like proteins (UBLs) play a key role in their recognition by the specific activating enzymes (E1s) to launch their transfer through the respective enzymatic cascades thus modifying cellular proteins. UB and Nedd8, a UBL regulating the activity of cullin-RING UB ligases, only differ by one residue at their C-termini; yet each has its specific E1 for the activation reaction. It has been reported recently that UAE can cross react with Nedd8 to enable its passage through the UB transfer cascade for protein neddylation. To elucidate differences in UB recognition by UAE and NAE, we carried out phage selection of a UB library with randomized C-terminal sequences based on the catalytic formation of UB∼NAE thioester conjugates. Our results confirmed the previous finding that residue 72 of UB plays a "gate-keeping" role in E1 selectivity. We also found that diverse sequences flanking residue 72 at the UB C-terminus can be accommodated by NAE for activation. Furthermore heptameric peptides derived from the C-terminal sequences of UB variants selected for NAE activation can function as mimics of Nedd8 to form thioester conjugates with NAE and the downstream E2 enzyme Ubc12 in the Nedd8 transfer cascade. Once the peptides are charged onto the cascade enzymes, the full-length Nedd8 protein is effectively blocked from passing through the cascade for the critical modification of cullin. We have thus identified a new class of inhibitors of protein neddylation based on the profiles of the UB C-terminal sequences recognized by NAE.
    PLoS ONE 08/2013; 8(8):e70312. DOI:10.1371/journal.pone.0070312 · 3.23 Impact Factor
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    • "The model generated for the Ubc12/Uba3 Cys domain interface reveals a largely complementary complex that would require the Nedd8 E1 UFD to rotate 17 further than observed in the doubly loaded Nedd8 E1 structure (Huang et al., 2007) (Figures 7B and S7C). Of note in the E2/Cys domain interface is Ubc12 Lys147 that corresponds to Pro121 in Ubc4, a Ubc4 residue that contacts the conserved Uba1-specific Cys domain hydrophobic patch (Figures 7E, 7F, and S3). "
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    ABSTRACT: Ubiquitin (Ub) conjugation is initiated by an E1 enzyme that catalyzes carboxy-terminal Ub adenylation, thioester bond formation to a catalytic cysteine in the E1 Cys domain, and thioester transfer to a catalytic cysteine in E2 conjugating enzymes. How the E1 and E2 active sites come together during thioester transfer and how Ub E1 interacts with diverse Ub E2s remains unclear. Here we present a crystal structure of a Ub E1-E2(Ubc4)/Ub/ATP⋅Mg complex that was stabilized by induction of a disulfide bond between the E1 and E2 active sites. The structure reveals combinatorial recognition of the E2 by the E1 ubiquitin-fold domain (UFD) and Cys domain and mutational analysis, coupled with thioester transfer assays with E1, Ubc4, and other Ub E2s, show that both interfaces are important for thioester transfer. Comparison to a Ub E1/Ub/ATP⋅Mg structure reveals conformational changes in the E1 that bring the E1 and E2 active sites together.
    Molecular cell 02/2013; 49(5). DOI:10.1016/j.molcel.2013.01.013 · 14.02 Impact Factor
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