Conservation of ligand-binding residues in VDR, PXR, and CAR. From high-resolution, x-ray crystallographic structures of human VDR, rat VDR, human PXR, mouse CAR, and human CAR bound to various ligands, the amino acid residues that directly interact with ligands are known (see Figure 5; also see Additional file 1: Genes used for phylogenetic analysis for complete list of species available and their accession numbers). (A) Of the 22 amino acid residues shown to interact with ligands at human and/or rat VDRs, only 4 residues show any sequence variation across vertebrate species. The remaining 18 of 22 residues show complete conservation across all vertebrate VDRs (from sea lamprey to human VDRs). Eighteen VDRs were used for the analysis. Due to partial sequence, data for the chimpanzee VDR was only available for the first two ligand-binding residues (corresponding to human VDR Y143 and F150); in addition, data was missing for the four most C-terminal ligand-binding residues (corresponding to human VDR H397, L414, V418, and F422) for crocodile, snake, turtle, lizard, frog, and fugu-β VDRs. (B) In contrast to the VDRs, the PXRs show extensive amino acid sequence divergence at the residues shown to interact with ligands at the human PXR. Only 3 of 23 positions are conserved throughout the 13 vertebrate PXRs while for 9 of 23 residues, over half of the PXRs have an amino acid residue that differs from that at the human PXR. Also indicated are the 9 amino acid residues in the BXRα and/or BXRβ lineages that show evidence for positive selection (see Figure 5 legend for more details; * indicates BXRα and/or BXRβ residue directly orthologous to human PXR ligand-binding residue; ** indicates residue adjacent to such a ligand-binding residue). (C) CARs also show much more divergence at ligand-binding positions than human VDR but not as great as the PXRs. The data is based on eight complete mammalian CAR sequences.