Figure 5. Homology model of hPKR1. The model is viewed perpendicular to the plasma membrane, with the extracellular side of the receptor shown on top, and the intracellular side shown on the bottom of the figure. The structure is colored from the N (blue) to the C (orange) terminal amino acid sequence. The insert shows the 7TM- bundle allosteric small-molecule binding site, predicted by the QSite Finder server. The binding site is located among TMs 3,4,5,6, and 7. doi:10.1371/journal.pone.0027990.g005 binding site capable of binding drug-like compounds, similar to the well-established TM bundle binding site typical of many family A GPCRs [25]. This is in addition to a putative extracellular surface binding site, which most likely binds the endogenous hPKR ligands, which are small proteins. Several synthetic small-molecule hPKR antagonists have been recently reported [17,18,19,20,38]. We hypothesized that these small molecules will occupy a pocket within the 7TM bundle [23,53]. To identify the potential locations of a small-molecule-TM binding site, we first mapped all receptor cavities. We then utilized two energy-based methods, namely, Q-SiteFinder [35] and SiteHound [36], to locate the most energetically favorable binding sites by scanning the protein structure for the best interaction energy with different sets of probes. The most energetically favorable site identified by the two methods overlaps; it is located in the upper part of the TM bundle, among TMs 3,4,5,6, and 7. The position of the identified pocket is shown in the insert in Figure 5. According to the structural superposition of the hPKR1 model on its three template structures, the predicted site is similar in position to the well-established TM-bundle binding site of the solved X-ray structures [54,55]. Furthermore, specific residues lining these pockets, which are important for both agonist and antagonist binding by GPCRs [25], are well aligned with our model (figure S2). Comparing the identified TM-bundle binding site between the two subtypes revealed that they are completely conserved, except for one residue in ECL2 - Val207 in hPKR1, which is Phe198 in hPKR2. Figure S5 presents a superposition of the two models, focusing on the binding site. This apparent lack of subtype specificity in the TM-bundle binding site is in agreement with the lack of specificity observed in activity assays of the small-molecule triazine-based antagonists [17], which could suppress calcium mobilization following Bv8 (a PK2 orthologue) stimulation to the same degree, in hPKR1 and hPKR2 transfected cells [17]. Small-Molecule Binders of Prokineticin Receptors We therefore will focus mainly on hPKR1 and will return to the issue of subtype specificity in the Discussion. Docking of known small-molecule antagonists to hPKR1 binding site and identification of important interacting residues To understand the mechanistic reasons for the need of particular pharmacophores for ligands activity, one has to look for interactions between the ligands and the receptor. As a preliminary step, we performed a validation study, aimed at determining whether our modeling and docking procedures can reproduce the bound poses of representative family A GPCR antagonist-receptor crystallographic complexes. We first per- formed redocking of the cognate ligands carazolol and cyano- pindolol, back to the X-ray structures from where they were extracted and from which the loops were deleted. The results indicate that the docking procedure can faithfully reproduce the crystallographic complex to a very high degree (figure S6 – A–C); with excellent ligand RMSD values of 0.89–1.2 A˚ between the docked pose and the X-ray structure (see table S2), in accordance with similar previous studies [44,56,57]. The redocking process could also reproduce the majority of heavy atomic ligand-receptor contacts observed in the X-ray complex and more generally, the correct interacting binding site residues and specific ligand- receptor hydrogen bonds, despite docking to loopless structures. Next, we built homology models of b1adr and b2adr and performed docking of the two antagonists into these models to examine the ability of homology modeling, combined with the docking procedure, to accurately reproduce the crystal structures. As can be seen from figure S6 and from the ligand RMSD values in table S2, the results can reproduce the correct positioning of the ligand in the binding site, and at least part of the molecule can be correctly superimposed onto the crystallized ligand, although the resulting RMSD values are above 2 A˚. The overall prediction of interacting binding site residues is good, correctly predicting 47– 66% of the interactions (see Table S2). We therefore performed molecular docking of the small- molecule hPKR antagonist dataset to the predicted hPKR1 allosteric 7TM-bundle binding site, to explore the possible receptor-ligand interactions. The set of 56 active and 51 inactive small-molecule antagonists was subjected to flexible ligand – rigid receptor docking to the hPKR1 model using LigandFit (as implemented in DS2.5, Accelrys, Inc.) [34]. For each compound the 50 best energy conformations were generated and docked into the binding site, resulting in an average of 250 docked poses for each molecule. The final ligand poses for each molecule were selected based on the highest LigScore1 docking score, since no experimental data regarding possible ligand contacting residues was available. The best scoring docking poses were analyzed visually for features that were not taken into account in the docking calculation, such as appropriate filling of the binding site – such that the compound fills the binding site cavity, and does not "stick out". Specific ligand-receptor interactions were monitored across all compounds. Figure 6 shows representative docked poses of two active (A,B) and two inactive compounds (C,D). As shown, the active molecules adopt a confirmation that mainly forms interactions with TMs 2, 3, and 6, such that the ligand is positioned in the center of the cavity, blocking the entry to it and adequately filling the binding site, as described. In contrast, the inactive small molecules are apparently incapable of simultaneously maintaining all of these contacts, and are positioned in different conformations that mostly maintain interactions with only some of the TMs mentioned. PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27990