Unnatural amino acids as tools to study protein phosphorylation and ubiquitylation

Abstract

Post-translational modifications (PTMs) of ubiquitin (Ub) have become more and more the focus of attention in recent years since they add a new layer of complexity to the ubiquitin code. Phosphorylation of Ub S65 (Ub pS65) is the most prominent PTM of Ub, but apart from its critical role in mitophagy, only little is known about the functions of Ub pS65. In this study, we use the method of genetic encoding of phosphoserine (pSer) and its non-hydrolyzable analog (nhpSer) to generate phosphorylated/ non-hydrolyzably phosphorylated Ub at either serine 20, 57 or 65. With these phosphorylated Ub variants in hands, we tested their influence on the different steps of the ubiquitin-conjugation cascade in comparison to unmodified Ub and Ub variants containing the widely used phosphomimic amino acids aspartate and glutamate. By this, we gained deeper insight into the process of pUb activation by the E1 enzyme UBA1 and show that Ub phosphorylation at S65 negatively affects the E3 ligase activity of E6AP and Hdm2. The site-by-site comparison of Ub pS65 and a mutant stabilizing the so-called C-terminally retracted conformation of Ub revealed that they show identical behavior on all three steps of the Ub cascade, indicating that the effects observed are caused by the retracted conformation of Ub pS65. The reported Parkin activation by Ub pS65 could also be confirmed. Furthermore, Ub nhpS65 performed similar to Ub pS65 in functional and structural analyses, which enabled us to make use of the non-hydrolyzable variant in the chase for phosphospecific Ub interactors. The ubiquitin-like modifier (UBL) NEDD8 is the closest relative of Ub. Both consist of 76 amino acids and harbor a serine residue at position 65 that is subject to phosphorylation. However, while the functional and structural consequences of S65 phosphorylation have been studied extensively for Ub, the respective features of NEDD8 remain widely enigmatic. We observed that like Ub, S65-phosphorylated NEDD8 (NEDD8 pS65) acts as an allosteric activator of the ubiquitin ligase Parkin and that phosphorylation induces an alternative NEDD8 conformation characterized by a retracted C-terminal tail. By using cell extracts and affinity enrichment coupled to mass spectrometry, we show that NEDD8 pS65 and Ub pS65 have different interactomes that are also distinct from those of the unmodified forms. Among the preferential NEDD8 pS65 interactors, members of the HSP70 family were identified and, indeed, functional studies revealed that NEDD8 pS65 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings provide evidence that phosphorylation of Ub and NEDD8 at S65 is involved in stress response signaling and underscore the importance of studying non-covalent interactions of NEDD8 in particular and post-translational modifications of UBLs in general.