Structural Basis for the Enzymatic Activity of the HACE1 HECT‐Type E3 Ligase Through N‐Terminal Helix Dimerization

Abstract HACE1 is an ankyrin repeat (AKR) containing HECT‐type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well‐known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo‐EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N‐terminal AKR domain, a middle (MID) domain, and a C‐terminal HECT domain. Its unique G‐shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N‐terminal helix of one monomer restricts access to the C‐terminal domain of the other. The in vitro ubiquitination assays, hydrogen‐deuterium exchange mass spectrometry (HDX–MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition.