Conclusions In conclusion, we have identified a small-molecule TM-bundle site that can accommodate the known small-molecule hPKR antagonists. Hence, it can be explored in the future for designing additional PKR-targeting compounds. The VLS procedure identified tens of compounds that are likely to affect hPKRs. Interestingly, FDA-approved drugs may also bind to these receptors, and in some instances, such as with Indinavir, this binding may provide a potential explanation for the drug’s side effects. One residue in ECL2 is different between the two subtypes (Val207 in hPKR1 corresponding to Phe198 in PKR2), and several residues in the intracellular loops may affect phosphory- lation. These residues may be exploited for designing subtype- specific pharmacological tools, to target different pathological conditions involving hPKRs. Supporting Information Figure S1 Structure-based multiple sequence alignment of modeled PKR subtypes and X-ray structures used as templates in the modeling procedure. Alignment was generated by the TCoffee server. The most conserved residue in each helix is shaded yellow and is indicated by its Ballesteros- Weinstein numbering [33]. Identical residues are in red and similar residues are in blue. bRho - bovine Rhodopsin (PDB code:1L9H), hB2ADR - human b2-adrenergic receptor (2RH1), hA2AR - human A2A adenosine receptor (3EML). The sequence of T4 lysozyme that was fused to the hB2ADR and hA2AR proteins to facilitate structure determination was removed prior to alignment, for clarity. (TIF) Figure S2 Structural superposition of the PKR1 model and GPCR X-ray templates used for homology model- ing. All structures are shown in ribbon representation. PKR1 is in turquoise, human b2-adrenergic is in orange (A), bovine rhodopsin is in gold (B) and human A2A-adenosine receptor is in gray (C). (D) Superposition of the hPKR1 model and the b2-adrenergic receptor structure with emphasis on the TM-bundle binding site. The structures are shown in a view looking down on the plane of the membrane from the extracellular surface. Binding site residues experimentally known to be important for ligand binding are denoted as sticks and are labeled with Ballesteros-Weinstein numbering. The T4 lysozyme fusion protein was removed from the b2-adrenergic and the A2A-adenosine receptor structures, for clarity. Structural superposition was performed using the Match- maker module in UCFS Chimera version 1.4.1. (TIF) Figure S3 Structures of the three known PKR antago- nists that were used as reference compounds for constructing ligand-based pharmacophore models. (TIF) Figure S4 Structural similarity between the identified VLS hits plotted as a heatmap. The degree of similarity was calculated using the Tanimoto coefficient, as described in Methods, and ranges between 0 (completely dissimilar com- References Small-Molecule Binders of Prokineticin Receptors pounds) and 1 (identical compounds). Compounds with similarity values .0.85 are usually considered structurally similar. Color intensity corresponds to the similarity value according to the legend. The heatmap was prepared using Matlab version 7.10.0.499 (R2010a). (TIF) Figure S5 Structural superposition of human PKR1 and PKR2 models. Both structures are shown in ribbon represen- tation, with hPKR1 in turquoise and hPKR2 in khaki. The insert shows a detailed view of the predicted transmembrane binding site, with side chains denoted as sticks. Structural superposition was performed using the Matchmaker module in UCFS Chimera version 1.4.1. (TIF) Figure S6 Predicted binding modes of cognate ligands redocked into crystal structures and homology models. (A) Cyanopindolol redocked to b1adr crystal structure (PDB code: 2VT4), (B) Carazolol redocked to b1adr crystal structure (2YCW), (C) Carazolol redocked to b2adr crystal structure (2RH1), (D) Cyanopindolol docked to b1adr homology model, (E) Carazolol docked to b1adr homology model and (F) Carazolol docked to b2adr homology model. The docked ligands are shown as green sticks. X-ray structures are represented as gray ribbons and the crystallized ligand is shown as gray sticks. In panels (D–F) the homology models are shown as gold ribbons. (TIF) Figure S7 Measure of Ka/Ks ratio on the amino acid sequence of the PKR subtypes suggests positive selection acting only on PKR2. Ka/Ks ratio (v) representing the ratio of non-synonymous (Ka) to synonymous (Ks) nucleotide substitution rates was calculated for each site for the PKR subtypes. The ratio is plotted against the amino acid position for hPKR1 (A) and hPKR2 (B). Residues showing v.1 are indicative of positive Darwinian selection, while residues showing v,1 are indicative of purifying selection; the ratio for neutral selection is one (indicated on the graph by a red line). Significant positive selection (p = 0.001) was detected only for PKR2, by the likelihood ratio test, and is concentrated in the N-terminus and C-terminus domains. (TIF) Potential hits identified from the ZINC Table S1 database. (DOC) Table S2 Ligand RMSD values and contact analysis for cognate ligand docking to b1adr and b2adr crystal structures and homology models. (DOC) Acknowledgments We thank Prof. Lior Wolf for helpful comments. Author Contributions Conceived and designed the experiments: AL MYN RM. Performed the experiments: AL. Analyzed the data: AL TY MYN. Contributed reagents/ materials/analysis tools: AW. Wrote the paper: AL MYN RM. 1. Kaser A, Winklmayr M, Lepperdinger G, Kreil G (2003) The AVIT protein family. 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PLoS ONE | www.plosone.org 15 November 2011 | Volume 6 | Issue 11 | e27990