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.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China.
Shan
S
He
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China.|Georgia Cancer Center, Augusta University, Augusta GA, USA.
Yu
Y
Cao
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China.
Ping
P
Xie
Department of General Surgery, Chinese People's Liberation Army General Hospital, Beijing 100853, China.
Guanglong
G
Dong
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Collaborative Innovation Center for Cancer Medicine, Beijing 100850, China.
Lingqiang
L
Zhang
D000595:Amino Acid Sequence; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002465:Cell Movement; D057809:HEK293 Cells; D006801:Humans; D062105:Molecular Docking Simulation; D009154:Mutation / Q000235:genetics; D000075687:NEDD8 Protein / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016212:Transforming Growth Factor beta / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D019485:Bone Morphogenetic Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D016212:Transforming Growth Factor beta; C498342:SMURF1 protein, human; C471641:SMURF2 protein, human; D044767:Ubiquitin-Protein Ligases