Several hot spot regions include more than one consensus sites. large number of potential arrangements within the binding site explains why PXR is able to accommodate a large variety of compounds. All five hot spots include at least one important residue, which is conserved in all mammalian PXRs, suggesting that the hot spot locations have remained largely invariant during mammalian evolution. The same side chains also show a high level of structural conservation across hPXR structures. However, each of the hPXR hot spots also includes residues with moveable side chains, further increasing the size variation in ligands that PXR can bind. Results also suggest a unique signal transduction mechanism between the PXR homodimerization interface and its co-activator binding site. The human pregnane X receptor, PXR, is a transcriptional regulator of a large number of genes involved in steroid and xenobiotic metabolism and excretion (1, 2), including Cholecalciferol cytochrome P450 (CYP) 3A4 (3, 4), CYP2B6 (5), aldehyde dehydrogenases, glutathione-S-transferase, sulfotransferases, and many others. Like other nuclear receptors, PXR contains both a DNA-binding domain and a ligand-binding domain. However, unlike the classical steroid, retinoid, and thyroid hormone receptors, which are highly selective for their cognate hormones, PXR has evolved to detect structurally diverse compounds. Human PXR activators include a wide range of prescription and herbal drugs such as paclitaxel, troglitazone, rifampicin, ritonavir, clotrimazole, and St. John’s Wort, which can be involved in clinically relevant drugCdrug interactions (6). Cholecalciferol ANPEP Cholecalciferol PXR can also be activated by various environmental chemicals, including polychlorinated biphenyls (7), phthalates (8), and xenoestrogens (9). PXR is also activated by pregnanes, androstanes, bile acids, hormones, dietary vitamins, and a wide array of other endogenous molecules (10). Although these diverse interactions imply promiscuity, PXR also exhibits Cholecalciferol specificity. A recent paper describing the use of machine learning methods for predicting human PXR activation is based on a training set of 98 activators and 79 non-activators, and tested on a nonoverlapping set of 82 activators and 63 non-activators (11). In some cases, activators differ from non-activators in only a few atoms. Thus, PXR binds diverse but precise arrays of compounds, a property defined as directed promiscuity (12). This fine-tuned mechanism of promiscuous and yet selective recognition is further evidenced by the substantial differences in the pharmacologic activation profile of PXR across species (12-15). Examples of promiscuous recognition are well documented in the literature (16-19), in most cases describing protein-protein or protein-peptide interactions. The promiscuous recognition of small molecules by proteins is probably best studied for hepatic mammalian cytochrome P450s (CYPs), which are able to metabolize a large variety of substances. Since many of these compounds are relatively recent synthetic products, their recognition by CYPs is clearly independent of molecular evolution and thus indicates genuine promiscuity, as in the case of CYP3A4, which metabolizes an estimated 50% of all clinically approved drugs (16). It is well established that the promiscuity of CYPs is largely due to the substantial plasticity of the CYP binding site (20, 21). In spite of an overall conserved architecture, CYP classes substantially differ in terms of the shape of the binding region. The core CYP structure has a number of loops that allow for substantial backbone flexibility, giving it a remarkable ability to accommodate ligands that differ in size, shape, and polarity, and frequently display non-Michaelis-Menten kinetics of substrate binding (19-22). The induced fit due to side chain motion is very important even for cases without a major conformational change (21, 22). We note that the Cholecalciferol promiscuity of binding observed in some protein-protein.