Selectivity of E2-E3 interactions in the human ubiquitin system

Abstract

Highly selective interactions between ubiquitin-conjugating enzymes and RING-type E3 ligases are crucial for the adequate and efficient action of ubiquitin and ubiquitin-like pathways. Within these cascades, E2 enzymes provide a connecting link between activation and the final covalent conjugation, thereby maintaining the integrity of the E1-E2-E3 hierarchic pyramid. It became increasingly clear that E2s are directly involved in specifying chain topology and thereby the fate of the substrate. Insights into the molecular basis of E2-E3 interaction specificity can direct future studies towards understanding ubiquitin network biology and associated diseases. Therefore, the studies described in this thesis aim at unraveling the selectivity of E2-E3 interactions in the human ubiquitin-proteasome system, both on the level of individual E2 and E3 proteins as well as their molecular determinants of interaction specificity. A global interaction screen intended to identify binary interactions between 35 human E2 UBC-folds and 250 human RING-finger domains is revealed 346 high-confidence interactions are in good agreement with literature-curated E2-E3 interactions and with GST pull-down verifications. As additional validation, E3-interaction interface of the K63-specific Ubc13(UBE2N) was changed by introducing two mutations so that its interface mimics that of the mixed-chain specific E2 UbcH5B(UBE2D2). Furthermore, this thesis provides an in-depth overview of the evolutionary constraints that influences protein organization, enzyme regulation and selectivity towards E1s and E3s of the E2 super family in light of ubiquitin and ubiquitin-like conjugation pathways. In addition, the relation between E2 sequence conservation and E3 selection was studied. The low number of E3 interactions of UbcH8(UBE2E2), with residue E66 in Helix 1 and D113 in Loop 1, could be rescued by mutation to the UbcH6(UBE2E1) counterpart residues (E66D and D113E), thereby mimicking the E3 interactions of UbcH6(UBE2E1). Using a combination of molecular dynamics simulations, protein threading and additional mutagenesis, models were generated between the UBC-folds of UbcH6(UBE2E1) and UbcH8(UBE2E2) with the RING-finger of TOPORS that explains how D113 is orientated in UbcH8(UBE2E2). Elongating the side chain of D113 by substitution with the glutamic acid of UbcH6(UBE2E1) enables UbcH8(UBE2E2) D113E to interact with the RING-finger of TOPORS, rescuing the shortness of D113 and thereby the interaction with TOPORS. Furthermore, an intramolecular network of salt-bridges of two strictly conserved residues (K117 and D145) that actively control the positioning of the D113 side-chains was studied. Taken together, research described in this thesis increase the knowledge of selectivity of crucial protein interactions in the ubiquitin system and opens new possibilities towards the development of small molecular compounds for cancer and ubiquitin-related diseases.