Although many eukaryotic proteins are N-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein, Nedd8, to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12’s N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12’s interactions with Dcn1, and prevents repulsion of a charged N-terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation, and define a mechanism for N-terminal acetylation-dependent recognition.

SUMMARY Most E3 ligases use a RING domain to activate a thioester-linked E2~ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2~UBL-target intermediate representing RBX1-UBC12~NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2~UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RINGE2~UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.

Summary SCF (Skp1-Cul1-Fboxes) E3 ligases are activated by ligation to the ubiquitin-like protein Nedd8, which is reversed by the deneddylating Cop9 Signalosome (CSN). However, CSN also promotes SCF substrate turnover through unknown mechanisms. Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCFSkp2/Cks1 and SCFFbw7 and found that CSN occludes both SCF functional sites – the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and inhibits SCF-catalyzed ubiquitin chain formation independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state, and inhibits a catalytically-competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination.

N-terminal acetylation is an abundant modification influencing protein functions. Since ≈80% of mammalian cytosolic proteins are N-terminally acetylated, this potentially represents an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions, suggesting it may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M, aka UBC12) and DCN1 (aka DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl amide binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress the anchorage-independent growth of a cell line harboring DCN1 amplification. Overall, the data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets, and provide insights into targeting multiprotein E2–E3 ligases.

Summary Little is known about molecular recognition of acetylated N-termini, despite prevalence of this modification among eukaryotic cytosolic proteins. We report that the family of human DCN-like (DCNL) co-E3s, which promote ligation of the ubiquitin-like protein NEDD8 to cullin targets, recognizes acetylated N-termini of the E2 enzymes UBC12 and UBE2F. Systematic biochemical and biophysical analyses reveal 40- and 10- fold variations in affinities amongst different DCNL-cullin and DCNL-E2 complexes, which contribute to widely ranging efficiencies of different NEDD8 ligation cascades. Structures of DCNL2 and DCNL3 complexes with N-terminally acetylated peptides from UBC12 and UBE2F illuminate a common mechanism by which DCNL proteins recognize N-terminally acetylated E2s, and how selectivity for interactions dependent on N-acetyl-methionine can be established through sidechains recognizing distal residues. Distinct preferences of UBC12 and UBE2F peptides for inhibiting different DCNLs, including the oncogenic DCNL1 protein, suggest it may be possible to develop small molecules blocking specific N-acetyl-methionine-dependent protein interactions.