A conserved mechanism of HECT E3-ligases regulation by auto-ubiquitylation

The attachment of ubiquitin (Ub) by HECT E3-ligases regulates numerus cellular process and consequently subjected to tight regulation. During the last ~20 years several highly specific mechanisms that regulate the HECTs activity by expression, phosphorylation or binding to inhibitory partners has been demonstrated for some of the HECTs. Recently, our lab discovered an auto-ubiquitylation based allosteric mechanism that regulates the activity of HECT ligases. We demonstrated that ubiquitylation on a conserved lysine residue located at the HECT alpha helix #1 ( α1) induces an allosteric mechanism that promotes oligomerization and inactivation of the ligase. The mechanism was showed with two HECT E3s belonging to the Nedd4 subfamily including yeast Rsp5 and human NEDD4-1. Here we show the conservation of the mechanism in the human HECT family members. Using x-ray crystallography and AlphaFold we predicted the position of the conserved lysin residues in all human HECT members. To assess these predictions, we mutated the HECT members in the putative lysine to arginine. We then compared the wild type and the mutants’ activities by a novel bacterial genetic selection system based on a split chloramphenicol resistance protein, recently developed in our lab. The linkage between the ligase activity and bacterial growth provides a simple readout for ubiquitylation. We found that the mutants present hyperactivity and exhibited a significant higher growth rate, suggesting that auto-ubiquitylation of these lysine residues regulate these HECTs.