Identification of molecular determinants required for interaction of ubiquitin-conjugating enzymes and RING finger proteins

Recent results from several laboratories suggest that the interaction of E2 ubiquitin-conjugating enzymes with the RING finger domain has a central ro le in mediating the transfer of ubiquitin to proteins. Here we present a mu tational analysis of the interaction between the E2 enzyme UbcM4/UbcH7 and three different RING finger proteins, termed UIPs, which, like Parkin, cont ain a RING1-IBR-RING2 motif. The results show that the E2 enzyme binds to t he RING1 domain but not to the other cysteine/histidine-rich domains of the RING1-IBR-RING2 motif. Three regions within the UbcM4 molecule are involve d in this interaction: the H1 alpha helix, loop L1, connecting the third an d fourth strand of the beta sheet, and loop L2, located between the fourth beta strand and the second alpha helix. Loop L2 plays an important role in determining the specificity of interaction. The effects of L2 mutations on UbcM4/UIP interaction are different for each UIP, indicating that RING fing er domains can vary considerably in their structural requirements for bindi ng to E2 enzymes. The result that single amino-acid changes can regulate bi nding of E2 enzymes to different RING finger proteins suggests a novel appr oach to experimentally manipulate proteolytic pathways mediated by RING fin ger proteins.