Distinct Liver X Receptor Alpha Residues at the Protein‐Protein Interface Mediate Ligand Dependent Transactivation in Heterodimeric Contexts

Liver X receptor alpha (LXRα) plays a critical role in the maintenance of lipid and cholesterol homeostasis. Ligand binding and dimerization with retinoid X receptor (RXR) or peroxisome proliferator‐activated receptor (PPAR) are required for forming active DNA binding complexes leading to gene regulation. Structure based prediction and solvent accessibility shows that residues H383, E387, H390, L414, and R415 located in helices 9 and 10 may be critical for protein‐protein interactions. To determine the contributions of residues to dimerization, ligand binding, and transactivation by the LXRα heterodimers, LXRα interface residues were individually mutated. The effects of each mutated LXRα on ligand binding, protein‐protein association, subcellular localization, and transactivation activity were investigated. Ligand binding studies showed that mutants L414R and R415A lack binding to T‐0901317, but retain binding to 25‐HC. Fluorescent protein‐protein binding assay demonstrated a decreased affinity of L414R for PPARα. Weak binding of L414R or R415A with PPARα did not alter protein conformations as determined by circular dichroism spectroscopy. Cell based bimolecular fluorescence complementation assay further confirmed the lack of binding of L414R to PPARα, but not to RXRα. Furthermore, L414R and R415A exhibit low levels of ligand dependent luciferase activity driven by the SREBP‐1c promoter. However, R415A shows higher basal levels of basal levels of luciferase activity when tested in the context of ApoA1 promoter. Taken together, our study demonstrates that charge reversal at the interface provides selectivity to dimerization, ligand binding, and plays a critical role in the ligand‐dependent transactivation activity of LXRα. Support or Funding Information This work was supported by USPHS NIH grant DK77573 to H.A. H. and funds from the Boonshoft School of Medicine and the College of Science and Mathematics( A ) Schematic representation of the LXRα domain structure showing single point mutations. ( B ) Contacts across the LXRα dimer interface. Location of amino acid residues H383, E387, H390, L414, and R415 in helices 9 and 10 across the LXRα‐RXRβ heterodimerEffects of mutations on dimerization of LXRα with PPARα assessed through fluorescent protein‐protein binding assay. Representative curves from fluorescence binding experiments are shown for binding of each LXRα mutant to PPARα. All mutants, except H383E, showed reduced binding affinity compared to the wild‐type. At least three independent experiments were performed for each analysisFar UV CD of the PPARα and LXRα proteins. Experimentally observed (Obs, open circles) circular dichroic spectrum of a mixture of 0.2 μM PPARα and 0.2 μM (A) wild‐type, (B) H383E, (C) E387Q, (D) H390E, (E) L414R, and ((F) R415A LXRα compared to the calculated average (Calc, closed circles) of the individually obtained PPARα and LXRα spectra representing non‐interacting proteins.Effects of LXRα interface mutations on ligand binding of (A) wild‐type, (B) H383E, (C) E387Q, (D) H390E, (E) L414R, and (F) R415A LXRα to T‐0901317. All mutants, except H383E, showed reduced binding affinity compared to the wild‐type. Three independent experiments were performed for each analysisVisualization of protein complexes composed of PPARα and (A) wild‐type, (B) H383E, (C) E387Q, (D) H390E, (E) L414R, and (F) R415A LXRα in living cells using BiFC analysis. Fluorescence images of COS‐7 cells expressing ECFP‐LXR, Venus‐PPAR, and Cerulean‐RXR proteins were acquired 24 hr after transfection.