Sphingosine 1-phosphate analogue recognition and selectivity at S1P4 within the endothelial differentiation gene family of receptors

Abstract

Synergistic computational and experimental studies provided previously unforeseen details concerning the structural basis of S1P (sphingosine 1-phosphate) recognition by the S1P4 G-protein-coupled receptor. Similarly to reports on the S1P1 receptor, cationic and anionic residues in the third transmembrane domain (R3.28 and E3.29 at positions 124 and 125) form ion pairs with the phosphate and ammonium of S1P, and alanine mutations at these positions abolished specific S1P binding, S1P-induced receptor activation and cell migration. Unlike findings on the S1P1 receptor, no cationic residue in the seventh transmembrane domain interacts with the phosphate. Additionally, two previously undiscovered interactions with the S1P polar headgroup have been identified. Trp186 at position 4.64 in the fourth transmembrane domain interacts by a cation-pi interaction with the ammonium group of S1P. Lys204 at position 5.38 forms an ion pair with the S1P. The S1P4 and S1P1 receptors show differences in binding-pocket shape and electrostatic distributions that correlate with the published structure-activity relationships. In particular, the binding pocket of mS1P4 (mouse S1P4) has recognition sites for the anionic phosphate and cationic ammonium groups that are equidistant from the end of the non-polar tail. In contrast, the binding pocket of hS1P1 (human S1P4) places the ammonium recognition site 2 A (1 A=0.1 nm) closer to the end of the non-polar tail than the phosphate recognition site.

Unique Ligand Selectivity of the GPR92/LPA5 Lysophosphatidate Receptor Indicates Role in Human Platelet Activation

“…This residue is also conserved in the S1P-EDG receptors and has been found to abolish ligand activation when mutated to alanine in S1P 1 and S1P 4 (16,39,40). Thus, Arg-3.28 is a critical residue for ligand recognition of both LPA and S1P EDG family receptors.…”

“…Lys-5.38 is conserved in the EDG family S1P receptors and has been shown to ion-pair with the phosphate of S1P in S1P 1 and S1P 4 , although it is essential for S1P recognition only in S1P 4 (36,40). In EDG family LPA receptors, 5.38 is an arginine in both LPA 2 and LPA 3 , and the residue ion-pairs with the phosphate of LPA, whereas an aspartate at this position in LPA 1 does not contribute to ligand activation (14,21).…”

“…We chose the LPA 1 model as a starting point because none of the GPCR for which crystal structures are available provides a higher degree of sequence homology. We have previously extensively evaluated the ligand recognition of LPA 1-3 and the S1P receptors, S1P 1 and S1P 4 , using computational model-guided mutagenesis (14,16,21,39,40), and we have identified a cluster of four nearly conserved amino acid residues critical for ligand-induced receptor activation. The residue at 3.28 is a positively charged arginine residue in all the EDG family of S1P and LPA receptors, and the adjacent residue at 3.29 is glutamic acid in all EDG-S1P receptors and glutamine in all EDG-LPA receptors.…”

Identification of the Hydrophobic Ligand Binding Pocket of the S1P1 Receptor

“…The Gln/Asn-3.29 residue also plays an essential role in ligand binding, because substitution to alanine results in a loss of S1P and LPA binding and receptor activation. We also succeeded in elucidating differences between S1P 1 and S1P 4 , as in the latter subtype Lys-5.38 and Trp-4.64 together compensate for the lack of a cationic residue at position 7.34 as in S1P 1 (27). These polar head group interactions are essential for ligand binding, activation, and specificity.…”

“…This effort has enabled us to identify S1P receptor residues that make essential interactions with the charged phosphate and amino moieties of the S1P pharmacophore. We have identified three basic amino acids, Arg-3.28, Lys-5.38, and Arg-7.34 in S1P 1 and S1P 4 that form salt bridges with the phosphate group of S1P and are essential for ligand binding in one or both receptors (26,27). Furthermore, we have pinpointed position 3.29, conserved as glutamine in LPA-and glutamate in S1P-specific members of the EDG family, as the single locus that determines ligand specificity for S1P versus LPA through required ion pairing between glutamate and the ammonium moiety of S1P (28).…”