Small-Molecule Binders of Prokineticin Receptors Figure 8. The essential activity-determining groups of the known binders form specific interactions with the receptor. (A) An example of the ligand’s chemical properties, which are important for its activity, identified in the SAR analysis, and how they interact with receptor residues. An active molecule is shown docked into the hPKR1 binding site. The activity-related chemical groups are indicated in red. The ligand is denoted by yellow sticks. Interacting receptor residues are shown as green sticks and labeled. Hydrogen bonds are denoted by dashed green lines, p- cation interactions are denoted by orange lines. (B) Schematic 2D representation of ligand-receptor interactions. The residues shown have at least one atom within 4A˚ of the ligand. Blue lines indicate hydrogen bonds and orange lines indicate hydrophobic interactions. Residues shaded in green are involved in van der Waals interactions. Residues involved in hydrogen bonds, charge, or polar interactions are shaded in cyan. doi:10.1371/journal.pone.0027990.g008 bitter taste receptors (summarized in [25]). This position is highly conserved within different family A GPCRs subfamilies, but it is divergent among these subfamilies, for example, an Asp in the aminergic receptors, compared with a Thr in hormone protein receptors. It was therefore assumed that the position may play a role in specific ligand binding within certain subfamilies [55]. Similarly, we suggest that although the residue type is divergent between the different subfamilies (for example, a positive Arg in the Prokineticin receptors compared with a negative Asp in aminergic receptors), its importance in ligand binding in such diverse receptors may be due to its spatial location in the TM- bundle binding site. In addition, Arg3076.58 is analogous to Tyr2906.58 of the GnRH receptor, which was found to be important for binding the GnRH I and GnRH II peptide ligands [63]. The equivalent residue at position 6.58 is also suggested, by mutagenesis studies, to play an important role in ligand binding and/or receptor activation of other peptide GPCRs, such as the NK2 tachykinin receptor [64], the AT1A angiotensin receptor [65], and the CXCR1 chemokine receptor [66]. Moreover, in the recent crystallographic X-ray structure of the CXCR4 chemokine receptor bound to a cyclic peptide antagonist, a specific interaction between position 6.58 and the peptide was observed [67]. Hence, position 6.58 may serve as a common position for the binding of both peptides (such as the endogenous ligands PK1 and PK2) and small-molecule ligands. Finally, in our analysis position 2.61, which is occupied by a Glutamic acid in hPKRs, was found to be essential for antagonist binding, since an electrostatic interaction may be formed between this negatively charged residue and the positive charge on the ligand. This may explain the need for the positive charge on the known small-molecule antagonists, which was indeed deduced from the structure-activity analysis. The ligand’s positive charge may interact with the negatively charged residue in receptor position 2.61, which was also shown to be important in ligand binding in the dopamine receptors [55]. In summary, the observed interactions reinforce the predicted putative binding site and may support the concept that family A GPCRs share a common small-molecule binding pocket inside the TM cavity, regardless of the nature of their cognate ligand. Docking of ligands to a single experimental or model structure of a GPCR receptor has been shown to reproduce the binding mode of the ligands in several cases [44,68,69], to enrich known PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e27990