Abstract
RING domains of E3 ligases promote transfer of Jib (ubiquitin) from the E2 similar to Ub conjugate to target proteins. In many cases interaction of the E2 similar to Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked TAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2 similar to Ub binding and Jib transfer. Mutation of the aromatic residue abolishes Jib transfer, but not interaction with Jib. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Jib and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.