Communication between the ERRalpha homodimer interface and the PGC-1alpha binding surface via the helix 8-9 loop

Area of Science:

• Molecular biology
• Structural biology
• Biochemistry

Background:

• Nuclear receptor (NR) and coactivator interactions are vital for gene regulation.
• Ligand-binding domain (LBD) structural studies reveal general binding modes but limited specificity determinants.
• Estrogen receptor-alpha (ERalpha) and estrogen-related receptor-alpha (ERRalpha) exhibit distinct coactivator binding preferences for PGC-1alpha.

Purpose of the Study:

• To elucidate the structural and functional basis for differential coactivator binding specificity between ERRalpha and ERalpha.
• To investigate the role of specific PGC-1alpha regions and ERRalpha structural elements in coactivator interaction.
• To understand how ERRalpha's helix 8-9 loop influences coactivator binding and allosteric regulation.

Main Methods:

• X-ray crystallography to determine the structure of the ERRalpha LBD complexed with a PGC-1alpha box3 peptide.
• Site-directed mutagenesis of PGC-1alpha and ERRalpha to assess the functional relevance of identified interaction sites.
• Comparative structural analysis between ERRalpha and ERalpha LBDs.
• Coactivator interaction assays using wild-type and mutant proteins.

Main Results:

• A novel crystal structure reveals interactions between ERRalpha LBD and PGC-1alpha residues N-terminal to the LXXYL motif (NR box3).
• These interactions involve helix 4, the helix 8-9 loop, and the C terminus of ERRalpha LBD, and are essential for efficient ERRalpha/PGC-1alpha binding.
• The helix 8-9 loop, differing significantly between ERRalpha and ERalpha, is identified as a key determinant of coactivator binding specificity.
• The helix 8-9 loop in ERRalpha allosterically links the LBD homodimer interface with the coactivator binding cleft.

Conclusions:

• ERRalpha exhibits unique binding interactions with PGC-1alpha, mediated by residues outside the canonical LXXLL motif.
• The ERRalpha helix 8-9 loop plays a critical role in conferring coactivator binding specificity and regulating interactions with PGC-1alpha.
• ERRalpha's helix 8-9 loop acts as an allosteric modulator, explaining distinct coactivator binding to ERRalpha monomers and homodimers.