Investigation of mixed ubiquitin chains and chain conformations
Structural Biology
Final Report Abstract
Ubiquitination is an essential posttranslational modification regulating many processes. So far ub research has focused primarily on the study of homogeneously linked polyubiquitin chains. However, there is growing evidence that chains containing more than one linkage type exist in vivo either in form of mixed-linkage-chains or branched chains. In this grant application we set out to investigate the involvement of the E2 enzyme Ubc1 and in particular of its UBA domain in the assembly of K48/K63-branched chains. Using mass spectrometry, we could detect proteomic changes indicating a function of Ubc1-facilitated ub branch formation in chromatin remodeling and in cellular stress response pathways in the context of DNA replication in S. cerevisiae. To identify proteins that bind to branched ub chains we performed a ub interactor screen. We were able to identify some of the first K48/K63 branched ubiquitin chain interactors, including huntingtin-interacting protein HIP1, E3 ligase UBR4 and DNA replication and repair factor RFC1. Finally, we developed an EPR based method and calculation protocol to determine the conformation space of ub covalenty bound to an E2 enzyme, in this case yeast Ubc7. In titration experiments we could show that the RING domain E3 ligase Doa10 leads to a reduction of the conformation space, pushing the complex towards a closed conformation. In contrast, the RING E3 ligase Hrd1 did not change the conformation space but accelterated ub transfer by allosteric modulation of acidic loop in the vicinity of the active site cysteine.
Publications
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The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains. The EMBO Journal, 40(6).
Pluska, Lukas; Jarosch, Ernst; Zauber, Henrik; Kniss, Andreas; Waltho, Anita; Bagola, Katrin; von Delbrück, Maximilian; Löhr, Frank; Schulman, Brenda A.; Selbach, Matthias; Dötsch, Volker & Sommer, Thomas
