Recombinant Expression of the HECTD1 Catalytic HECT Domain for Biochemical and Crystallographic Studies

Abstract

Ubiquitination is a post-translational modification that regulates virtually every cellular process within eukaryotes. Ubiquitin can be conjugated to a substrate as a single moiety or in the form of polyubiquitin chains. These chains can be assembled through 8 different linkages between two ubiquitin molecules, resulting in a wide diversity of polyubiquitin chain architectures. Intriguingly, recent studies have revealed that atypical mixed and branched polyubiquitin chains confer specific functions in eukaryotic cells, such as enhanced proteasomal degradation. E3 ubiquitin ligases mediate the conjugation of ubiquitin to target substrates, with the HECT sub-family of E3 ligases coordinating this process via their catalytic HECT domain. The HECTD1 E3 ubiquitin ligase has recently been identified to assemble branched atypical K29/K48 polyubiquitin chains. However, the mechanisms that regulate its catalytic activity and chain specificity are poorly understood.
This research focused on elucidating the structural elements of the catalytic HECTD1 HECT domain to determine how they regulate the formation of branched K29/K48 polyubiquitin chains. Multiple sequence alignments highlighted three unique regions within the HECTD1 HECT domain that were absent in other HECT family members. The absence of these regions in the homologous S. cerevisiae ancestor UFD4, suggested these regions evolved to facilitate HECTD1’s ability to assemble branched K29/K48 polyubiquitin chains. An optimised HECTD1 HECT domain recombinant protein was expressed and purified for crystallographic studies, resulting in the formation of crystals that diffracted to ~3.5 Å resolution. The HECTD1 HECT domain was additionally revealed to form oligomers during its purification. Using a range of biophysical techniques such as SAXS-SEC-MALLS, monomeric, dimeric and tetrameric HECTD1 HECT domain species were confirmed, with subsequent in vitro autoubiquitination assays highlighting the tetrameric conformation as the most catalytically active. Furthermore, chemically crosslinked dimeric and tetrameric HECTD1 HECT domain species were purified. The conserved α1-helix located at the N-terminus of all HECT E3 HECT domains, was found to promote higher oligomerisation and aggregation of the HECTD1 HECT domain. Interestingly, pseudo phosphorylation of this helix relieved this effect, alongside mildly reducing HECTD1 catalytic activity, indicative of a phosphorylation dependent regulatory mechanism for the oligomerisation and catalytic activity of the HECTD1 HECT domain.
Overall, this research provides a foundation for future crystallographic and Cryo-EM studies investigating the catalytic HECT domain of HECTD1. Furthermore, the discovery of the three unique regions found within the HECTD1 HECT domain, alongside the role of the α1-helix in regulating its oligomerisation has provided new avenues for future research aimed at investigating the regulation and determinants of HECTD1’s polyubiquitin chain specificity.

Date of Award	17 Jan 2022
Original language	English
Awarding Institution	University of Bath
Sponsors	Alzheimer's Research UK
Supervisor	Julien Licchesi (Supervisor) & Robert Williams (Supervisor)