Unconventional Ubiquitin E2 enzymes and their activation by E3 ligases

• Applicant: Dr. Tobias Ritterhoff
• Subject Area: Biochemistry ; Structural Biology
• Term: from 2016 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 329595285

Posttranslational modification of proteins with Ubiquitin is an essential and extremely conserved process in eukaryotic cells that is involved in numerous physiological and disease pathways. Ubiquitin E3 ligases, of which RING-type E3 ligases represent the vast majority, have traditionally been viewed as specific regulators of this process. They act by regulating Ubiquitin E2 conjugating enzymes, which, during modification, form a thioester bond with Ubiquitin and can transfer it onto a lysine in a target protein. Importantly, since RING E3s allosterically activate lysine reactivity of E2s and thus do not chemically participate in modification, it is the E2s that determine the product of the reaction, i.e. at what residue of the target protein ubiquitylation takes place and whether it is mono- or polyubiquitylation. Thus, a thorough molecular understanding of all the roughly 40 different E2 enzymes is crucial. Recent years have witnessed the identification of E2s that are able to transfer Ubiquitin to reactive groups other than the amino function of lysines. Paradoxically, both, these unusual and the canonical, lysine-reactive E2s, use a conserved catalytic domain, which despite its small size (~17 kDa) appears to be able to accommodate an incredible catalytic diversity. Here, I propose to study two of the largely uncharacterized E2s with unconventional reactivity: (1) Ube2J2, which was reported to transfer Ub to serine and threonine in addition to lysine residues and (2) Ube2W, which is specific with the N-alpha amino group of target protein N-termini. My structural and biochemical analysis of these two enzymes will help to uncover how their active sites can accommodate their unusual specificity and how they are mechanistically regulated by their cognate RING E3 ligases. In addition, the relevance of my molecular findings will be assessed in vivo. Ultimately, this work will lead to a more comprehensive understating of the molecular aspects and significance of the non-lysine ubiquitylation.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Rachel Klevit