Mechanism of non-canonical ubiquitination by ubiquitin conjugating enzyme Ubc6
by Anuruti Swarnkar
Date of Examination:2022-10-20
Date of issue:2023-10-18
Advisor:Dr. Alexander Stein
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Dirk Görlich
Referee:Dr. Sonja Lorenz
Referee:Dr. Alex Caspar Faesen
Referee:Prof. Dr. Henning Urlaub
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EnglishProtein quality control by ubiquitination occurs due to the sequential action of three enzyme classes: ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligases (E3). Conventionally, ubiquitin (Ub) attachment by these enzymes occurs via isopeptide bonds, typically to the ε-amino group of lysine residues. Seminal studies from the past have established that non-lysine residues such as serine, threonine and cysteine can also be ubiquitinated via hydroxy- and thio-ester bonds. However, the molecular basis and physiological relevance for such non-canonical ubiquitination remain elusive. Among the E2 family, yeast Ubc6 and its homologs are the only E2 enzymes known to mediate serine/threonine ubiquitination via hydroxyester linkages, but the structural or the chemical determinants for hydroxyl attachment of Ub have not been identified. To investigate the mechanism of hydroxyester ubiquitination, I have characterized Ubc6 using structural studies and in vitro biochemical assays with purified protein. I found that Ubc6 lacks conserved motifs and residues involved in ubiquitin transfer in other E2s. Instead, Ubc6 contains a highly conserved insertion proximal to its active-site Cys (C87), consisting of Ser (S89) and His (H94). The in vitro data suggest that the conserved histidine 94 (H94) within this insertion imparts reactivity towards hydroxylated amino acids. Substitution of H94 with methylated histidine showed that H94 has to exist in its neutral, non-protonated form to exert this activity. This suggests that H94 acts as a general base by abstracting a proton from the incoming hydroxyl group. However, it cannot be formally excluded that H94 acts by covalent catalysis. Moreover, I have described a crystal structure of Ubc6 in a Ub bound state which shows that the conserved serine 89 (S89) plays a role in stabilizing a reactive closed-state of Ubc6. Mutation of S89 led to a loss of E3 RING mediated activation of Ubc6 activity, suggesting that the closed conformation is disfavoured by mutating S89. In addition, in vitro assays showed that the active site of Ubc6 is selective towards serine over threonine indicating a preference towards primary hydroxylated nucleophiles. Overall, my results indicate that conserved residues in the active site proximal region of Ubc6 mediate reactivity towards hydroxylated amino acids, filling a gap in our understanding of the chemical determinants of this process. In the second part of this thesis, I investigated the functional relevance of a conserved C-terminal element (CTE) in Doa10. Doa10 is an E3 ligase involved in the conserved protein quality control pathway termed endoplasmic reticulum associated protein degradation (ERAD). During ERAD, membrane and luminal misfolded proteins are ubiquitinated and retrotranslocated back into the cytosol where they are degraded by the proteasome. The ERAD machinery also recognizes soluble and membrane bound cytoplasmic misfolded proteins, majorly by the Doa10 E3 ligase complex. However, the structural elements within Doa10 that allow recognition of cytoplasmic misfolded proteins are not known. Using co-immunoprecipitation assays combined with proteomics, I found a role of CTE in the ubiquitin ligase activity of Doa10. However, such an involvement was not recapitulated using in vitro assays, most likely due to absence of well-defined substrates. This thesis provides chemical and structural insights into the mechanism of hydroxyester ubiquitination by Ubc6. This could potentially serve as a reference for understanding non-canonical ubiquitination mediated by other E2/E3 enzymes.
Keywords: Protein quality control; Non-canonical hydroxyester linked ubiquitination; E2 catalytic activity; In vitro ubiquitin discharge assay