Analysis of tractable allosteric sites in G protein-coupled receptors

Abstract

Allosteric modulation of G protein-coupled receptors represent a promising mechanism of pharmacological intervention. Dramatic developments witnessed in the structural biology of membrane proteins continue to reveal that the binding sites of allosteric modulators are widely distributed, including along protein surfaces. Here we restrict consideration to intrahelical and intracellular sites together with allosteric conformational locks, and show that the protein mapping tools FTMap and FTSite identify 83% and 88% of such experimentally confirmed allosteric sites within the three strongest sites found. The methods were also able to find partially hidden allosteric sites that were not fully formed in X-ray structures crystallized in the absence of allosteric ligands. These results confirm that the intrahelical sites capable of binding druglike allosteric modulators are among the strongest ligand recognition sites in a large fraction of GPCRs and suggest that both FTMap and FTSite are useful tools for identifying allosteric sites and to aid in the design of such compounds in a range of GPCR targets.

Figure 1

Experimentally validated allosteric sites in GPCRs. As reference shown is a Class A orthosteric antagonist ligand in grey CPK with protein in yellow ribbon (Adenosine A_2A, triazine ligand PDB:3UZA) then from bottom to top: Intracellular Class A antagonist for CCR9 (vercinon ligand in orange CPK, PDB:5LWE); Extra-helical Class A ago-PAM for GPR40 (ligand AP8 in fuchsia CPK, PDB:5TZY); Extra-helical Class A inverse agonist for complement C5a (NDT9513727 ligand in light green CPK, PDB:5O9H); Extra-helical Class B allosteric antagonist GCGR (MK0893 ligand in pink, PDB:5EE7); Allosteric Class B antagonist CRF1 (CP376395 ligand in brown CPK, PDB:4K5Y); Extra-helical Class A antagonist for PAR2 (AZ3451 ligand in dark grey CPK, PDB:5NDZ); Allosteric Class C NAM for mGlu5 (M-MPEP in cyan CPK, PDB: Extra-helical Class A antagonist P2Y₁ (BPTU ligand in green CPK, PDB:4XNV); Intra-helical Class A allosteric partial agonist (MK-8666 ligand in lilac, PDB:5TZR); Intra-helical Class A allosteric agonist (TAK-875 ligand in purple, PDB:4PHU); Allosteric Class A antagonist for PAR2 (AZ8838 ligand in blue CPK, PDB:5NDD).

Figure 2

Hot spots and allosteric ligand binding sites predicted by (a) FTMap and (b) FTSite for PDB 5X7D. Also shown are the hot spots and orthosteric ligand binding site by (c) FTMap and (d) FTSite. We note that in this and all following figures, each probe cluster is represented by the structure of a single probe at the cluster center. Both ligands are represented by green sticks. The FTMap hot spots, shown as lines, are coloured by rank in the following order: cyan, hot pink, yellow, light pink, white, blue and orange. The FTSite sites, shown as mesh, are coloured, by rank, in the following order: pink, green, and purple.

Figure 3

Hot spots and ligand binding sites predicted, respectively, by (a) FTMap and (b) FTSite for the mGluR5-mavoglurant structure (PDB: 4OO9) and by (c) FTMap and (d) FTSite for the mGluR5-HTL14242 structure (PDB: 5CGD). The allosteric ligand mavoglurant is represented by green sticks. The FTMap hot spots are shown as lines, 1(11) in yellow and 4(6) in green. The second ranked site, predicted by FTSite, is shown as green mesh. The allosteric ligand HTL14242 is represented by green sticks. HOH4115 is represented by a blue sphere. The FTMap hot spots, shown as lines, are colored as follows: 3(8) in white and 7(5) in teal. The third ranked FTSite site is shown as purple mesh.

Figure 4

Hot spots and ligand binding sites predicted, respectively, by (a) FTMap and (b) FTSite for the GPR40-TAK-875 structure (PDB: 4PHU) and by (c) FTMap and (d) FTSite for the GPR40-AP8 structure (PDB: 5TZY). The allosteric ligand TAK-875 is represented by green sticks. The FTMap hot spots, shown as lines, are 2(13) in light pink and 3(10) in white. The third ranked FTSite site is shown as purple mesh. The allosteric ligand AP8 is represented by green sticks. The FTMap hot spots, shown as lines are 0(16) in pink, 3(9) in white and 9(5) in yellow. The third ranked FTSite site is shown as purple mesh.

Figure 5

Hot spots and ligand binding sites predicted, respectively, by (a) FTMap and (b) FTSite for the CCR9-vercirnon structure (PDB: 5LWE). The allosteric ligand vercirnon is represented by green sticks. The FTMap hot spots, shown as lines, 1(13) in yellow, 2(13) in light pink, 4(11) in blue and 5(6) in orange. The highest ranked FTSite site is shown by pink mesh.

Figure 6

Hot spots and ligand binding sites predicted by FTMap and by FTSite for the orthosteric complexes of (a) beta2 (PDB:2RH1), (b) M2 (PDB:4MQS), (c) GPR40 (PDB:5TZR) and (d) P2Y1 (PDB:4XNW) receptors. The allosteric ligands are represented by green sticks. The FTMap hot spots, shown as lines, are coloured are coloured by rank in the following order: cyan, hot pink, yellow, light pink, white, blue and orange. The FTSite sites, shown as mesh, are coloured, by rank, in the following order: pink, green, and purple.