Identification of determinants in E2 ubiquitin-conjugating enzymes required for hect E3 ubiquitin protein ligase interaction

Members of the hect domain protein family are characterized by sequence sim ilarity of their C-terminal regions to the C terminus of EG-AP, an E3 ubiqu itin-protein ligase, An essential intermediate step in E6-AP-dependent ubiq uitination is the formation of a thioester complex between EG-AP and ubiqui tin in the presence of distinct E2 ubiquitin-conjugating enzymes including human UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several hect domain proteins, including Saccharomyces cerevisiae RSPB, form ubiqui tin thioester complexes, indicating that hect domain proteins in general ha ve E3 activity. We show here, by the use of chimeric E2s generated between UbcH5 and other E2s, that a region of UbcH5 encompassing the catalytic site cysteine residue is critical for its ability to interact with E6-AP and RS P5. Of particular importance is a phenylalanine residue at position 62 of U bcH5 that is con served among the members of the UBC4/UBC5 subfamily but is not present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interact ions. The conservation of this phenylalanine residue throughout evolution u nderlines the importance of the ability to interact with hect domain protei ns for the cellular function of UBC4/UBC5 subfamily members.