Unconventional Ubiquitin E2 enzymes and their activation by E3 ligases
Structural Biology
Final Report Abstract
Ubiquitylaton is one of the most versatile and consequential posttranslational protein modifications and essential in all eukaryotic cells. Modification of target proteins with the small protein Ubiquitin (Ub) is mediated by an enzymatc cascade at the center of which are class of enzymes called E2 conjugatng enzymes. During ubiquitylaton, E2s are conjugated with an actvated Ub molecule through a thioester bond and are then able to discharge it onto the lysine residue of a target protein (aminolysis reacton). For most ubiquitylaton reactons, discharge is regulated and provided specificity by so called RING E3 ligases. Over the past several years, a canonical picture of Ub discharge from an E2~Ub thioester conjugate has emerged, where its reactvity towards a target lysine correlates with the formaton of a non-covalent interacton between the E2 and its conjugated Ub, dubbed the closed conformaton. While in the conjugate alone, the Ub is very flexible with respect to the E2, the binding of a RING E3 ligase biases the conjugate towards the closed conformaton thus triggering discharge. This process is allosterically regulated through a key residue in the RING E3, dubbed the allosteric linchpin, which engages in hydrogen bond with both the E2 and its conjugated Ub. Of the about 40 human E2 enzymes, a handful has been described that can modify side chain residues other than lysine thus seemingly expanding the bandwidth of ubiquitylaton signalling. Because the only well-studied non-canonical E2, UbcH7, which can only discharge Ub to Cys residues, does not rely on the closed conjugate conformaton for actvaton, the mechanisms behind the actvaton of other non-canonical E2 enzymes are fascinatng and have been addressed in this work: I analyzed Ubc6, a hydroxy-reactve E2, and Ube2W, an E2 that modifies the Na amino group of proteins' flexible N-termini. Unexpectedly, I found that both E2s rely on a closed conjugate conformaton for their unusual reactvity. In the case of Ube2W, I found that its flexible C-terminus, which the enzyme uses to bind a substrate N-terminus, helps to restrict the flexibility of the conjugated Ub towards the closed conformaton. This effect is augmented when a substrate N-terminus is bound Ube2W C-terminus. In the case of Ubc6, I found that the conjugate naturally populates the closed conformaton to a high degree, which is surprising given that canonical key residues important for the closed conformaton interacton with Ub are not conserved in Ubc6. I could show that this propensity of the Ubc6 conjugate renders the linchpin residue of its cognate E3 ligases dispensable for actvaton. A functonally relevant, direct contact between Ub and the E3, in which Ub can only engage, when the conjugate binds to the E3 in the closed conformaton, seems sufficient for actvaton. As all published E2-Ub/E3 structures show similar direct Ub/E3 contacts, it appears that for most (canonical) E2s these contacts are a common and crucial aspect of actvaton as well, with the notable difference that acton by the E3's linchpin in these cases is absolutely necessary. Thus, I postulate the following model of E2 actvaton by RING E3 ligases: the linchpin's interacton with the E2 and Ub is the first step of an actvaton process, which helps to slightly decrease Ub's flexibility. This restricton then works in concert with the direct Ub/E2 and Ub/E3 contacts to help the conjugate sustainably assume the closed conformaton and allow discharge. In the case of Ubc6, the linchpin-mediated restricton of Ub's flexibility is achieved by its natural predispositon for the closed conformaton thus circumventng this level of regulaton. In sum, the findings obtained during this work not only provided new insight into the specific E2 enzymes studied, but produced a more detailed picture of E2 actvaton in general.
Publications
- A clickable glutamine (CliQ) derivatve for the traceless reversible modificaton of peptdes and proteins. Chem Commun (Camb). 2019 Feb 12;55(14):2043-2045
Whedon SD, Parker MK, Tyson EL, Ritterhoff T, Shelton PMM, Chatterjee C
(See online at https://doi.org/10.1039/c8cc09404g)
