Fig. 3. The PPARγ-E379K mutation alters interaction with RXRα.

a Structure analysis and computational modeling the crystal structure of an PPARγ:RXRα complex (PPARγ in red; RXRα in gray) bound to DNA using the HADDOCK2.2 web server shows a complex interaction network involving PPARγ-E379 and -K382 (LBD) and RXRα-Y189 and -K175 (DBD) in the WT complex (left panel). PPARγ-E379K alters the configuration of this interface (middle panel). Double charge reversal mutations in PPARγ (E379K) and RXRα (K175E) can restore the PPARγ LBD-RXRα DBD interface through a novel electrostatic interaction (right panel) (PDB entry 3DZY). Amino acid residues involved in the PPARγ LBD-RXRα DBD interface are indicated in the stick format. The figures were generated by PyMOL Molecular Graphics System Version 1.8 (2015) provided by SBGrid⁶⁰. b Spectroscopic analyses of helix 6 peptides. Left panel: ¹H,¹³C-HSQC spectra of PPARγ-WT^376–385 (red) and -PPARγ-E379K^376–385 (green) recorded in 20 mM Na₂HPO₄ /NaH₂PO₄ (pH 7.4) at 25 °C and overlaid. Signals originate from C^α. Middle panel: The C^α secondary chemical shifts (SCSs) for both the WT peptide (red) and the E379K variant (green)⁴⁹. In the WT peptide, Arg378–Leu381 showed consecutive positive Cα SCSs, indicating transient helical structure (gray box). Right panel: Far-UV CD spectra of PPARγ-WT^376–385 (red) and PPARγ-E379K^376–385 (green) recorded at 25 °C in 20 mM Na₂HPO₄/NaH₂PO₄ (pH 7.4). Dashed vertical line indicated the minimum at 222 nm for α-helix structure. c HEK293T cells were transiently cotransfected with expression vectors encoding WT or mutant PPARγ, WT or mutant RXRα, and the Lpl PPRE-minimal promoter-reporter, in the absence or presence of 1 µM rosiglitazone. Activation of the reporter is expressed as fold induction over empty vector (control). Data are presented as mean values + SEM, with individual data points indicated with circles, n = 3 biologically independent experiments. One-way ANOVA with Tukey’s multiple comparisons were used to compare cells transfected with mutant vs. WT; *p < 0.05. d Overexpression of the different PPARγ and RXRα proteins in HEK293T cells, as assessed by western blot analyses using a PPARγ- or RXRα- specific antibody. The arrows indicate PPARγ or RXRα, and the asterisk indicates an unknown non-specific band. Control, empty vector control; WT, wild-type. Three independent experiments were performed, and similar results were obtained. Source data for panel b–d are provided in the Source Data file.