Structural and functional studies on the interaction network of E2 ubiquitination enzymes

Abstract

Protein modification by ubiquitin conjugation (ubiquitination) is responsible for degradation of most cellular proteins, and therefore is crucial to most aspects of eukaryotic cells. Numerous diseases, including hypoxia, inflammatory diseases, muscle wasting disorders and cancer, were discovered to be affected by aberrations in the ubiquitin system. Ubiquitination is a highly regulated system carried out by complex processes through the hierarchical E1-E2-E3 enzyme cascade, which involves thousands of enzymes and substrates in human cells. The detailed molecular mechanisms underlying these complex processes, the specificity of E1-E2, E2-E3, and E3-substrate recognition are poorly understood. In this thesis, we describes the protein production of E3 RING domain, and structural and functional studies on the interaction network of E2 conjugating enzymes: the complexes of the E3 ligase c-Cbl RING with the E2 enzyme UbcH5b and that with UbcH7, and the complexes of the E2 Rad6b with the RING E3 Rad18 and ubiquitin. The E2 ubiquitin conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. We found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B~Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7~Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required, but not sufficient for transfer of ubiquitin to potential targets. In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyze either monoubiquitination or ubiquitin chain formation. Conjugation enzymes Rad6/Rad18 only monoubiquitinate PCNA to signal for ‘error prone’ DNA damage tolerance. Ubiquitin chain formation on PCNA is catalyzed by a different set of conjugation enzymes: Ubc13/MMS2/Rad5. Here we studied the structure of E3 Rad18 RING-RING symmetric dimer determined by X-ray, and the Rad18 RING dimers can bind two Rad6b E2 enzymes. In contrast, binding studies of full-length Rad18 homodimer with Rad6b show that Rad18 homodimer can bind only to a single Rad6b enzyme. Till now, the functional role of this asymmetry E3 dimers found in not only heterodimeric E3 ligases but also the homodimeric U-box E3 CHIP and RING E3 Rad18 is poor understood. Furthermore, we show that E2 enzyme Rad6 is intrinsically capable of ubiquitin chain formation. The specificity of the reaction is provided by E3 enzyme Rad18, which prevents the chain-formation activity to direct the reaction towards monoubiquitination. The R6BD of Rad18 provides the inhibition by competing with ubiquitin for a non-covalent ‘backside’ binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process.