N-terminal acetylation of eukaryotic proteins is an important and widespread modification, but its impact on protein-protein interactions is poorly understood. The E2 ubiquitin-conjugating enzyme Ubc12 cooperates with the RING E3 ubiquitin ligase Rbx1 and a co-E3, Dcn1, to mediate transfer of the ubiquitin-like protein Nedd8 to substrate cullins. Dcn1 interacts with an N-terminal extension on Ubc12, and Schulman and colleagues now show that NatC-catalyzed N-terminal acetylation of the extension enhances the interaction between Dcn1 and Ubc12, promoting neddylation of substrates. The crystal structure of an N-terminally acetylated Ubc12 peptide bound to Dcn1 shows the acetyl group making direct interactions with residues lining a hydrophobic pocket in Dcn1. Additionally, the acetyl group enhances the Ubc12-Dcn1 interaction by masking the positive charge at the end of the N-terminal Ubc12 a-helix; mutations in Dcn1 that can accommodate Ubc12's positive charge suppress the need for acetylation in vitro and in vivo. A stapled-helix Ubc12 peptide strengthens the Dcn1-Ubc12 interaction, indicating that conformational flexibility of Ubc12 also affects the affinity between the two proteins. Exploring the effects of N-terminal acetylation on other pathways will give further insights into the mechanisms by which this modification regulates diverse cellular processes. The authors propose that disrupting N-terminal acetylation-mediated interactions may have therapeutic potential in many systems. (Science doi:10.1126/science.1209307, published online 22 September 2011)