Regulation of ubiquitin transfer by XIAP, a dimeric RING E3 ligase

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RING domains of E3 ligases are essential for ubiquitin (Ub) transfer from E2~Ub conjugates to target proteins.
• E2~Ub conjugate interaction with RING domains frequently necessitates prior RING domain dimerization for E3 ligase activity.

Purpose of the Study:

• To investigate the role of RING domain dimerization in the E3 ligase activity of Inhibitor of Apoptosis (IAP) proteins, specifically XIAP and cIAP2.
• To elucidate the structural and biochemical basis of E2~Ub conjugate binding and ubiquitin transfer mediated by IAP RING domains.

Main Methods:

• Cross-linking experiments to identify interaction sites between E2~Ub conjugates and IAP RING domains.
• Structural and biochemical analyses of XIAP RING dimer to determine the role of specific residues in ubiquitin transfer.

Main Results:

• E2 conjugated ubiquitin was shown to contact the RING homodimer interface of XIAP and cIAP2.
• An aromatic residue at the XIAP RING dimer interface is critical for both E2~Ub binding and subsequent ubiquitin transfer.
• Mutation of this aromatic residue abrogated ubiquitin transfer, while interaction with ubiquitin remained intact, highlighting the importance of precise contacts.

Conclusions:

• Precise contacts between ubiquitin and the RING domain, particularly at the dimer interface, are essential for nucleophilic attack on the thioester bond.
• Unlike cIAP proteins where RING dimerization is an activation step, the XIAP RING domain forms a stable dimer, enabling constitutive E3 ligase activity without a separate activation step.