Structural studies of E1-E2 interactions in the ubiquitin cascade

Thesis

The conjugation of ubiquitin to substrate proteins underpins nearly all eukaryotic cellular pathways. In vertebrates, this process is initiated by two essential ubiquitin-activating enzymes (E1s), UBA1 and UBA6. Recent studies have shown that each E1 directs independent downstream pathways through distinct ubiquitin-conjugating enzymes (E2s), yet the mechanisms governing E2 selection remain unclear.

Here, in vitro E2 activity screens combined with cryoEM structures of the two UBA6-specific E2s in complex with UBA6 provide new insight into the molecular interactions driving E1-E2 specificity in the ubiquitin cascade. This framework was then applied to generate E2 mutants with altered E1 specificities, offering potential tools to dissect E1-specific pathways in cells.

Additionally, this work identifies a unique priority mechanism for the UBA6-specific E2 BIRC6, enabled by a uniquely high affinity E1-E2 interaction. Further analysis revealed that the Cys-cap loop of UBA6 dampens E2 affinities, which allows UBA6 to function with a high affinity E2 without being inhibited. These findings outline an affinity-driven hierarchy among E2s, balanced by an E1 regulatory element to ensure efficient E2 turnover.

Overall, this work provides key mechanistic insights into how E1-E2 specificity is achieved in the ubiquitin system, highlighting both structural determinants and regulatory mechanisms that shape pathway selectivity. By uncovering unique features of UBA6-mediated interactions and identifying principles that govern E2 hierarchy, these findings not only expand our fundamental understanding of the ubiquitin cascade but also lay the groundwork for new molecular tools to probe and manipulate E1-specific cellular pathways.

Authors: Riechmann, C

• DOI: 10.5287/ora-xq2jqo4q5
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Subjects: Biochemistry