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Abstract

Nedd4-family ubiquitin ligases (E3s) play a key role in several signal transduction pathways. Hence, addressing which mechanisms regulate their activity under normal conditions is of high relevance, considering they are promising targets for drug discovery. Although Nedd4 ligases exhibit high levels of conservatio, diverse regulatory mechanisms have been proposed within the family. The activity of various Nedd4 E3s, such as Smurf2 and Nedd4, is controlled through an auto-inhibitory interaction of the N-terminal C2 domain with the catalytic HECT domain. Using our recently develop NMR approach “methionine scanning” we have characterized the C2 domain-binding surface of the Smurf2 HECT domain, which partially overlaps with a noncovalent ubiquitin binding surface (UBS). Our in vitro ubiquitination assays and pull-downs show that he overlap of the C2 and the UBS interferes directly with the enzyme’s activity. Point mutations in the C2-HECT binding surface were able to release the C2-mediated auto-inhibition. In addition, structural models show that the conformation adopted by the full length protein does not allow for the transfer of Ub form the E2 to the HECT domain. These results also supported by our transthiolation assays. Lastly, analogous results were obtained for Nedd4, validating the same auto-inhibitory mechanism. Although sequence conservation between Smurf1–Smurf2 is higher than 80%, previous studies do not support auto-inhibition as the mechanism responsible for Smurf1 regulation. Using the aforementioned NMR approach we were able to detect an interaction between the C2 and HECT domain of Smurf1, what lead us to investigate whether auto-inhibition is indeed not conserved in Smurf1.