Identification of the Hydrophobic Ligand Binding Pocket of the S1P1 Receptor

Yuko Fujiwara,Daniel A Osborne,Michelle D Walker,De-An Wang,Debra A Bautista,Karoly Liliom,James R Van Brocklyn,Abby L Parrill,Gabor Tigyi

doi:10.1074/jbc.m609648200

Abstract

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.

Highlights

Introduction of charged residues to replaceM3.32K, L3.43E/ L3.44E, and L5.51E severely disrupted activation and either abolished or significantly reduced (L5.51E) ligand binding compared with the wild type (WT) receptor
We found six mutations that showed no or greatly reduced liganddependent activation yet maintained [32P]Sphingosine 1-phosphate (S1P) binding similar to the wild type (WT) S1P1
We replaced many of these residues with charged amino acids of similar size to probe whether disruption to hydrophobicity in the putative binding pocket would have an impact on receptor function

Summary

The abbreviations used are

S1P, D-erythro-sphingosine-1-phosphate; FACS, fluorescence-activated cell sorting; GPCR, G protein-coupled receptor; GTP␥S, guanine-␥-thiotriphosphate; TM, transmembrane domain; WT, wild type; EDG, endothelial differentiation gene; LPA, lysophosphatidic acid; BSA, bovine serum albumin. Our groups have embarked on a computational modeling-driven mutagenesis approach to delineate agonist recognition by S1P1 at the atomic level 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 succeeded in elucidating differences between S1P1 and S1P4, 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 S1P1 [27] These polar head group interactions are essential for ligand binding, activation, and specificity. We set out to identify the residues of S1P1 that interact with the aliphatic part of S1P, which we designate the hydrophobic binding pocket of the receptor. Ogous positions in other S1P receptors suggests modifications that will lead to selective agonists of each receptor

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION