Nonspecific yet decisive: Ubiquitination can affect the native‐state dynamics of the modified protein

Abstract

Ubiquitination is one of the most common post-translational modifications of proteins, and mediates regulated protein degradation among other cellular processes. A fundamental question regarding the mechanism of protein ubiquitination is whether and how ubiquitin affects the biophysical nature of the modified protein. For some systems, it was shown that the position of ubiquitin within the attachment site is quite flexible and ubiquitin does not specifically interact with its substrate. Nevertheless, it was revealed that polyubiquitination can decrease the thermal stability of the modified protein in a site-specific manner because of alterations of the thermodynamic properties of the folded and unfolded states. In this study, we used detailed atomistic simulations to focus on the molecular effects of ubiquitination on the native structure of the modified protein. As a model, we used Ubc7, which is an E2 enzyme whose in vivo ubiquitination process is well characterized and known to lead to degradation. We found that, despite the lack of specific direct interactions between the ubiquitin moiety and Ubc7, ubiquitination decreases the conformational flexibility of certain regions of the substrate Ubc7 protein, which reduces its entropy and thus destabilizes it. The strongest destabilizing effect was observed for systems in which Lys48-linked tetra-ubiquitin was attached to sites used for in vivo degradation. These results reveal how changes in the configurational entropy of the folded state may modulate the stability of the protein's native state. Overall, our results imply that ubiquitination can modify the biophysical properties of the attached protein in the folded state and that, in some proteins, different ubiquitination sites will lead to different biophysical outcomes. We propose that this destabilizing effect of polyubiquitin on the substrate is linked to the functions carried out by the modification, and in particular, regulatory control of protein half-life through proteasomal degradation.

Ubiquitylation Directly Induces Fold Destabilization of Proteins

“…2 and SI, Figure S6). This discussion is consistent with a recent computational study, which showed that a ubiquitylation-induced increase in entropy in the folded state of the substrate protein causes thermodynamic destabilization20. Furthermore, the induction of more anisotropic molecular motion by ubiquitylation may be a possible factor that causes these changes in the structural dynamics of the substrate proteins.…”

Non-degradative Ubiquitination of Protein Kinases

“…Hagai and Levy note that the thermodynamic effects of ubiquitination vary depending on the attachment site [ 118 ]. Atomistic MD simulations also show that ubiquitination of Ubc7, an E2 enzyme, decreases the flexibility of certain regions of Ubc7 although there were no specific direct interactions between ubiquitin and the E2 [ 120 ]. We observe similar stabilization of the C-helix with ubiquitination at K476, also in the absence of any stable interactions between ubiquitin and ZAP-70.…”

“…We observe similar stabilization of the C-helix with ubiquitination at K476, also in the absence of any stable interactions between ubiquitin and ZAP-70. The Ubc7 simulations also show that the strongest effect occurred with K48-linked tetraubiquitination at known sites of degradative ubiquitination [ 120 ], indicating that polyubiquitination could have an even larger effect on ZAP-70 conformation than monoubiquitination.…”

Releasing the Lockdown: An Emerging Role for the Ubiquitin-Proteasome System in the Breakdown of Transient Protein Inclusions

“…A couple of arguments assert that ubiquitin conjugates must be removed during triage: 1. Ubiquitin conjugation influences protein folding dynamics, thwarting the possibility of appropriate refolding [55]. 2.…”