RING dimerisation drives higher-order organisation of SINA/SIAH E3 ubiquitin ligases

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• RING-type E3 ubiquitin ligases are crucial for ubiquitylation.
• SIAH1 utilizes a catalytic RING domain and a substrate-binding domain (SBD).
• Previous studies indicate SBD-mediated homodimerization in SINA/SIAH ligases.

Purpose of the Study:

• To determine the crystal structure of the SIAH1 RING domain.
• To investigate the role of RING domain dimerization in SIAH1 E3 ligase activity.
• To elucidate the higher-order assembly of SINA/SIAH E3 ligases.

Main Methods:

• X-ray crystallography of the human SIAH1 RING domain.
• Static light scattering to validate dimerization in solution.
• Cellular localization studies using fluorescently tagged proteins.
• Co-localization assays with aggregated synphilin-1A.

Main Results:

• The crystal structure revealed a potential RING dimer of human SIAH1.
• RING dimerization was confirmed in solution and enhances SIAH1 E3 ligase activity in vitro and in cells.
• SIAH1, SIAH2, and SINA form cytoplasmic clusters, which are disrupted upon disabling RING dimerization.
• Wild-type SIAH1, but not its dimerization mutant, colocalizes with aggregated synphilin-1A.

Conclusions:

• RING domain dimerization is a key feature of SIAH1 E3 ligase function.
• Alternating RING:RING and SBD:SBD interactions likely organize SINA/SIAH proteins into higher-order homomultimers.
• SIAH1 multimerization may contribute to its substrate preference for aggregated proteins.