There are 28 unique members of the homologous to E6AP C-terminus (HECT) E3 ubiquitin ligase family responsible for catalytically transferring a ubiquitin moiety onto a specific substrate. This family contains a relatively conserved C-terminal HECT domain that mediates its ubiquitylation activity with a highly conserved cysteine residue. Of these, HECT Domain and Ankyrin Repeat Containing E3 Ubiquitin Protein Ligase 1 (HACE1) is one of the ‘other’ HECT E3 ubiquitin ligases containing an N-terminal ankyrin repeat domain responsible for facilitating substrate recognition and binding. Isolation of a biochemically pure HECT domain has proven difficult due to chronic insolubility and/or inhibited enzymatic activity. Recent in-silico bioinformatic analyses coupled with secondary structure prediction software indicated a conserved amphipathic α-helix preceding the N-termini in all HECT E3 ubiquitin ligases. This α -helix is proposed to bind to a hydrophobic pocket found within the HECT domain. Building on recently published structural studies and in-house biochemical and biophysical experiments, we show a drastic increase in protein yield, solubility, stability, and activity of the HECT domain of HACE1. This finding also holds true for other members of the HECT E3 ubiquitin ligase family. Our cumulative findings support a call to redefine the boundaries of the HECT domain to include this N-terminal extension that will likely be critical for future biochemical, structural, and therapeutic studies on the HECT E3 ubiquitin ligase family.
Support or Funding Information
This research was supported through Clark University and the National Institutes of Health, R15GM126432.
The solved HECT domain model AREL1 in comparison to its namesake E6AP. The bilobal structure of the HECT domain is shown with the C-lobe containing the catalytic cysteine (light grey), the E2-binding N-lobe (dark grey), and the N-terminal α-helical extension shown in AREL1 (magenta).
