Browsing Chemistry & Biochemistry Theses and Dissertations by Author "Abeykoon, Dulith Maduwantha Bandara"
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- ItemCHARACTERIZATION OF THE STRUCTURE AND BINDING OF BRANCHED K6/K48-LINKED AND BRANCHED K6/63-LINKED POLYUBIQUITIN CHAINS(2021) Abeykoon, Dulith Maduwantha Bandara; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ubiquitin (Ub) is an important post-translational protein modifier in eukaryotes. Post-translational modification with Ub is an essential process for eukaryotic cellular signaling including protein degradation, DNA repair, antigen-peptide generation and endocytosis. This post-translational modification with Ub occurs through ubiquitination where Ub attach as monoUb or polyUb chains. Ub form polyUb chains by forming covalent linkages between the C-terminus of one Ub and any of seven lysines or the N-terminus of other Ubs. Polyubiquitin chains can form homogeneous, heterogeneous, linear or branched chains, leading to diversity in polyubiquitin chain signaling outcomes. This diversity in signaling is due to the variety of conformations that arise based on the linkage specificity of the polyUb chains. Recently, branched K6/K48-linked polyubiquitins were shown to enhance deubiquitinating activity of UCH37 in the presence of Rpn13. To better understand the underlying structural mechanisms, here we determined the NMR structures of branched K6/K48-linked triubiquitin (Ub3) and discovered a previously unobserved interdomain interface between each of the distal ubiquitins and the proximal domain. We performed NMR binding assays to study the interactions of branched K6/K48-linked Ub3 with hHR23a UBA2, Rap80 tUIM and UCH37/Rpn13 complex. Binding studies of branched K6/K48-linked Ub3 to the UBA2 domain of the proteasomal shuttle protein hHR23A resulted in negligible differences between branched K6/K48-linked Ub3 and related dimers (K6-Ub2 and K48-Ub2). Interestingly, introducing hydrophobic patch surface residue mutations led to stronger affinity with both distal domains suggesting a change in the binding mode. Stronger binding affinity for K6/K48-linked branched Ub3 was observed with Rap80 tUIM. Moreover, deubiquitinating enzyme UCH37 (with Rpn13) showed strong affinity for both K6-linked and K48-linked distal domains, thereby suggesting a functional impact of this interdomain interface towards enhanced deubiquitinating activity of UCH37. Moreover, mutation studies of the hydrophobic patch residues of the proximal ubiquitin have shown the importance of the hydrophobic patch surface to maintain the interdomain interface of this branched trimer and for interactions with binding partners. Finally, initial studies done with the regulatory domain of the DNA repair protein p53 (p53c) have shown that p53c is a promising candidate for ubiquitination via non-enzymatic ubiquitination method introduced by our lab.