Mechanistic basis for ubiquitin modulation of a protein energy landscape

Abstract

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other non-degradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity--and, by extension, the myriad effects of ubiquitination on substrate proteins--arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrates C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Destabilization, however, appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a non-destabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single post-translational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality-control properties. (248 words) Significance StatementFluctuations on a protein energy landscapes encode the mechanistic basis for vital biological processes not always evident from static structures alone. Ubiquitination, a key posttranslational modification, can affect a proteins energy landscape with consequences for proteasomal degradation, but the molecular mechanisms driving ubiquitin-induced energetic changes remain elusive. Here, we systematically characterize the energetic effects of ubiquitination at three sites on a model protein. We find that distinct thermodynamic mechanisms can produce the same outcome of ubiquitin-induced destabilization at sensitive sites. At a non-sensitive site, we observe formation of a substrate-ubiquitin interaction that may serve to protect against destabilization. This work will enable development of predictive models of the effect of ubiquitin at any given site on a protein with implications for understanding and engineering regulated ubiquitin signaling and protein quality control in vivo.