Functional Interactions of E2~Ubiquitin Conjugates in and outside the Ubiquitination Pathway

Abstract

Thirty years of research have implicated ubiquitin (Ub) signaling in nearly every aspect of eukaryotic cell biology. What appears to be a simple signaling molecule in fact has the ability to encode countless cellular fates that include proteasomal degradation, cell cycle control, and DNA damage repair. Although the enzymes within the Ub transfer pathway are well established (E1, E2, E3), the molecular details required for protein ubiquitination are largely unknown. To address questions regarding the final stages of Ub transfer, we began with structural characterization of the pathway intermediate, the E2~Ub conjugate (~ indicates the thioester linkage between the E2 active site cysteine and the Ub C-terminus). NMR and SAXS techniques were used to describe the array of conformations populated by the flexibly linked UbcH5c~Ub and Ubc13~Ub conjugates. Unlike the Ubc13~Ub conjugate, the UbcH5c~Ub intermediate strongly prefers extended conformations in which the Ub is positioned directly below the E2 active site. We determined that addition of RING/U-box E3 ligases induced a shift in the ensemble of UbcH5c~Ub populated states toward more "closed" conformations. A variety of biochemical assays were used to show that these closed conformations represent the activated states by which E3 ligases promote Ub transfer. This mechanism of conformational activation is utilized by diverse RING/U-box E3:E2 pairs, and is mediated by an intermolecular hydrogen bond stemming from a conserved basic residue on the E3. To identify mutations that augment the E3's ability to facilitate Ub transfer, we collaborated with the Stan Fields lab to screen nearly 100,000 E3 sequence variants for ubiquitination activity. Rare mutations were identified that increased ligase activity as much as 20-fold. NMR analysis showed that the mutations fall into two classes based on their effects; the first class of mutants enhance activity by increasing E3:E2 binding affinity while the second class does so by augmenting the ability to induce closed E2~Ub conformations. Lastly, we characterized an interaction between the E2~Ub conjugate and Shigella effector kinase OspG to find that, while OspG has little effect on ubiquitination activity, formation of the OspG:UbcH5c~Ub complex results in a pronounced increase in OspG kinase activity.