Engineering the orthogonal ubiquitin E1-E2 pairs for identification of K27 chain linkage substrates

Ubiquitination is one kind of crucial protein post-translational modification (PTM) in eukaryotic cells, forming sorts of types of polyubiquitin chain linkages on the substrates. Ubiquitin conjugating enzymes (E2s) play an essential role in the formation of ubiquitin chains. However, the mechanism of the formation of atypical chain (such as K27) and the identification of the related substrates are not well understood. Previously we developed an orthogonal ubiquitin transfer (OUT) pathway to identify the substrates of a specific ubiquitin ligase (E3). In OUT pathway, a ubiquitin mutant (xUb) is transferred to the substrates through an engineered xE1-xE2-xE3 cascade. In this study, we reengineered a new OUT pathway for the transfer of xUb-K27, an isoform of xUb with only one lysine at Lys27 resident. The newly designed xUba1-xUbe2D2 (xE1-xE2) pairs can transfer xUb-K27 to downstream wild type E3s and form K27 linkages. Ube2D2 is known for its versatility as it forms all sorts of polyubiquitin chains on substrates. Therefore, the xE1-xE2 pairs are empowered to transfer other ubiquitin mutants, for instance, xUb-K6 and xUb-K11. The new xE1-xE2 pairs also deepen the understanding of structural information about the E1-E2 interaction, and provide further insights into the mechanism of chain formation mediated by E2.