Structural basis for PPARγ transactivation by endocrine-disrupting organotin compounds

Abstract

Organotin compounds such as triphenyltin (TPT) and tributyltin (TBT) act as endocrine disruptors through the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway. We recently found that TPT is a particularly strong agonist of PPARγ. To elucidate the mechanism underlying organotin-dependent PPARγ activation, we here analyzed the interactions of PPARγ ligand-binding domain (LBD) with TPT and TBT by using X-ray crystallography and mass spectroscopy in conjunction with cell-based activity assays. Crystal structures of PPARγ-LBD/TBT and PPARγ-LBD/TPT complexes were determined at 1.95 Å and 1.89 Å, respectively. Specific binding of organotins is achieved through non-covalent ionic interactions between the sulfur atom of Cys285 and the tin atom. Comparisons of the determined structures suggest that the strong activity of TPT arises through interactions with helix 12 of LBD primarily via π-π interactions. Our findings elucidate the structural basis of PPARγ activation by TPT.

Figure 1. Structures of the PPARγ-LBD complex with TBT (A) and TPT (B).

Figure 2. The organotins ((A): TBT, (B): TPT) and Cys285 in the ligand-binding pocket of PPARγ.

Anomalous difference electron density maps contoured at 3.5σ (red) indicate the position of the tin atom and omit 2F_O–F_C electron density maps contoured at 0.5σ (cyan) indicate the geometry of the aliphatic or aromatic chain. Distances between the tin and sulfur atoms are indicated. In panel (B), the major and minor conformations of TPT are shown in yellow and gray, respectively.

Figure 3. Interactions of PPARγ-LBD with TBT (A) or TPT (B).

Ligands are shown as cyan and yellow sticks. Ligand-interacting residues, which are close (within 4.2 Å) to the ligands, are also shown. Common interacting residues for both ligands are shown as gray sticks. Interacting residues for TBT or TPT only are show in green or pink, respectively.

Figure 4. Cell-based transcriptional activation assay of rosiglitazone, TPT, and TBT on wild-type, C285A, and F363A mutants of PPARγ.

Data are expressed relative to the levels of vehicle-treated cells; these levels were set to 1. Results are expressed as means ± 1 S.D. of three independent cultures. *P < 0.05 indicates values significantly different between 2 groups analyzed by using 2-way ANOVA.

Figure 5. Mass spectrometry of the PPARγ-LBD complex with TPT (A–C) or TBT (D, E) under non-denaturing conditions.

Mass spectra show that PPARγ forms a complex with TPT (C) or TBT (E) in a 1:1 molar ratio. Mass patterns after the addition of aliquots of formic acid (A, D = 3%, B = 1%) to the complex indicate that the dissociation of the interaction is caused by the unfolding of PPARγ-LBD.

Figure 6. The π-π interactions in the PPARγ-LBD/TPT complex.

The distances between the nearest neighbor carbon atoms of the aromatic rings are indicated.