Phosphorylation of PPARγ Affects the Collective Motions of the PPARγ-RXRα-DNA Complex

Abstract

Peroxisome-proliferator activated receptor-γ (PPARγ) is a nuclear hormone receptor that forms a heterodimeric complex with retinoid X receptor-α (RXRα) to regulate transcription of genes involved in fatty acid storage and glucose metabolism. PPARγ is a target for pharmaceutical intervention in type 2 diabetes, and insight into interactions between PPARγ, RXRα, and DNA is of interest in understanding the function and regulation of this complex. Phosphorylation of PPARγ by cyclin-dependent kinase 5 (Cdk5) has been shown to dysregulate the expression of metabolic regulation genes, an effect that is counteracted by PPARγ ligands. We applied molecular dynamics (MD) simulations to study the relationship between the ligand-binding domains of PPARγ and RXRα with their respective DNA-binding domains. Our results reveal that phosphorylation alters collective motions within the PPARγ-RXRα complex that affect the LBD-LBD dimerization interface and the AF-2 coactivator binding region of PPARγ.

Fig 1. Components of the PPARγ-RXRα-DNA ternary complex.

Protein structural domains (DBD, DNA-binding domain and LBD, ligand-binding domain) are indicated. Yellow spheres in the two DBD are Zn²⁺ ions. DNA is shown as a cartoon.

Fig 2. Interpolation of PPARγ structures along the sum of the first 7 eigenvectors from PCA for the holo complex, with insets for each of the other complexes studied here, focusing on the LBD.

Cyan indicates completely overlapping regions and thus little or no motion. Red and yellow areas represent those that are more mobile. Critical secondary structure elements are labeled on the holo complex and those features manifesting the greatest movement in each non-holo complex are indicated in the insets.

Fig 3. Movement of the H2’-H3 loop and the BVT.13 ligand in PPARγ complexes.

Positions of the loop in snapshots from the (a) holo complex and (b) phospho complex (red) simulations, (c) heavy-atom RMSD distributions of the BVT.13 ligand in both the holo and phospho complexes, from data pooled over all frames in all trajectories, and (d) representative conformations of the BVT.13 partial agonist in holo (blue) and phospho (red) complexes. In panels (a) and (b), one structure was taken from each of the three replicate simulations to indicate the three different states (indicated by different shades of red and blue). Helices H2 and H3 are labeled, as is the position of Ser245/pSer245. The “crystal” positions of the ligands in panel (d) are from the energy-minimized, equilibrated structures, with hydrogen atoms removed for clarity.

Fig 4. Backbone RMSF of (a) RXRα and (b) PPARγ DBD residues.

RMSF was measured from a trajectory pooled from the final 400 ns of each replicate simulation, for a total of 1.2 μs of sampling. A least-squares fit of backbone atoms in each DBD was performed prior to calculating the RMSF.

Fig 5. The PPARγ-RXRα LBD-LBD interface.

Labels in blue correspond to structural features of PPARγ, while those in red correspond to RXRα. At left is the full complex, including the PPRE DNA sequence. At right is a zoomed-in view of the LBD-LBD interface.

Fig 6. Free energy surfaces of interfacial dynamics of all complexes studied here.

Three free energy minima are identified in the holo complex simulations, and are labeled by Roman numerals. Images corresponding to representative holo complex conformations of each basin are shown, with the conformations being positioned most closely to the respective basins to which they correspond. Interfacial PPARγ and RXRα helices are in blue and red, respectively, and are labeled in the image nearest to Basin I. Positions along eigenvector 1 (x-axis) and eigenvector 2 (y-axis) are shown in nm for each free energy surface.

Fig 7. Characterization of PPARγ-RXRα tertiary structure dynamics at the LBD-LBD interface of the holo complex.

(a) The twist angle θ between the two subunits, and distances (b) r₁ and (c) r₂. In panels (a—c), structural elements of PPARγ and RXRα are in blue and red, respectively, and are labeled in each panel to indicate the relative orientation. Panel (d) is the free energy surface from PCA, enlarged from Fig 6, with basins labeled. Panels (e) and (f) show the distributions of r₂ and r₁, respectively, in all three basins. Panel (g) shows the distributions of θ in all three basins. Note the x-axes in panels (e) and (f) are reversed to more clearly correspond to the properties of the basins, as described in the text. The legend in the bottom-right referring to the basins is applicable to panels (e—g).