The Nedd8 Non-covalent Binding Region in the Smurf HECT Domain is Critical to its Ubiquitn Ligase Function

Nedd8 is a ubiquitin-like protein that controls vital biological events through conjugation to target proteins. We previously identified the HECT-type ubiquitin ligase Smurf1 which controls diverse cellular processes is activated by Nedd8 through covalent neddylation. However, the effect of non-covalent binding to Nedd8 remains unknown. In this study, we demonstrate that both Smurf1 and its homologue Smurf2 carry a non-covalent Nedd8-binding site within its catalytic HECT domain. Structural analysis reveals that Smurf2 has Nedd8-binding sites within the small sub-domain of N-lobe and the C-lobe of HECT domain. Interestingly, the consensus Nedd8 binding sequence, L(X7)R(X5)F(X)ALQ is conserved in both Smurfs. Mutational studies reveal that all the five residues in the conserved sequence are required for binding to Nedd8. Functional studies suggest that mutations that disrupt Smurf interaction with Nedd8 reduce its neddylation and stabilize the protein. Furthermore, Nedd8 binding site in Smurf is shown to be necessary for its ubiquitin ligase activity towards the substrate and also the self-ubiquitylation. Finally, we show that Nedd8 binding to Smurf plays important roles in the regulation of cell migration and the BMP and TGFβ signaling pathways.