REVEALING THE STRUCTURAL AND FUNCTIONAL PROPERTIES OF NON-CANONICAL K6-LINKED POLYUBIQUITIN CHAINS.

Abstract

Ubiquitin is an important protein modifier in eukaryotes, which tags the proteins and signals them to different pathways in the cell. It has been shown that polyubiquitin chains of different linkages act as distinct cellular signals. Non-canonical K6-linked polyubiquitin chains have been linked to breast and ovarian tumor suppressor protein BRCA1. However, detailed structural and binding studies of these chains had been hampered by the absence of an efficient way to synthesize them. We developed and optimized both non-enzymatic and enzymatic methods to synthesize K6-linked polyubiquitin chains. Dynamics of K6-linked diubiquitin chains were studied by solution NMR and ensemble analysis. We determined that K6-linked diubiquitin is present in at least of two conformations in solution. The conformers we determined from the analysis of solution data suggested the structural ability of K6-linked diubiquitin to bind protein receptors from both proteasomal degradation signaling pathway (hHR23a UBA2) and DNA Damage Repair pathway (Rap80 tUIM). In order to elucidate the pathway it is involved in, we performed NMR binding assays to determine which of these proteins K6-linked diubiquitin binds. We found that this diubiquitin binds both Rap80 tUIM and hHR23a UBA2 with a high affinity, suggesting that it has the functionality to be part of both DNA repair pathway and proteasomal signaling pathway. Thus, our studies with K6-linked diubiquitin have revealed that due to their conformational heterogeneity, these chains have the capability to be part of both proteasomal degradation signaling and DNA damage repair pathways.