Identification of the hydrophobic ligand binding pocket of the S1P 1 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

Research output: Contribution to journalArticle

46 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2374-2385
Number of pages12
JournalJournal of Biological Chemistry
Volume282
Issue number4
DOIs
StatePublished - Jan 26 2007

Fingerprint

Lysosphingolipid Receptors
Ligands
G-Protein-Coupled Receptors
Computer Simulation
Chemical activation
Small Molecule Libraries
Amino Acids
Mutagenesis
Sphingolipids
Drug Design
National Institutes of Health (U.S.)
Second Messenger Systems
sphingosine 1-phosphate
Linings
Intercellular Signaling Peptides and Proteins
Screening
Genes
Crystal structure
Health
Mutation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Fujiwara, Y., Osborne, D. A., Walker, M. D., Wang, D. A., Bautista, D. A., Liliom, K., ... Tigyi, G. (2007). Identification of the hydrophobic ligand binding pocket of the S1P 1 receptor. Journal of Biological Chemistry, 282(4), 2374-2385. https://doi.org/10.1074/jbc.M609648200

Identification of the hydrophobic ligand binding pocket of the S1P 1 receptor. / Fujiwara, Yuko; Osborne, Daniel A.; Walker, Michelle D.; Wang, De An; Bautista, Debra A.; Liliom, Karoly; Van Brocklyn, James R.; Parrill, Abby L.; Tigyi, Gabor.

In: Journal of Biological Chemistry, Vol. 282, No. 4, 26.01.2007, p. 2374-2385.

Research output: Contribution to journalArticle

Fujiwara, Y, Osborne, DA, Walker, MD, Wang, DA, Bautista, DA, Liliom, K, Van Brocklyn, JR, Parrill, AL & Tigyi, G 2007, 'Identification of the hydrophobic ligand binding pocket of the S1P 1 receptor', Journal of Biological Chemistry, vol. 282, no. 4, pp. 2374-2385. https://doi.org/10.1074/jbc.M609648200
Fujiwara Y, Osborne DA, Walker MD, Wang DA, Bautista DA, Liliom K et al. Identification of the hydrophobic ligand binding pocket of the S1P 1 receptor. Journal of Biological Chemistry. 2007 Jan 26;282(4):2374-2385. https://doi.org/10.1074/jbc.M609648200
Fujiwara, Yuko ; Osborne, Daniel A. ; Walker, Michelle D. ; Wang, De An ; Bautista, Debra A. ; Liliom, Karoly ; Van Brocklyn, James R. ; Parrill, Abby L. ; Tigyi, Gabor. / Identification of the hydrophobic ligand binding pocket of the S1P 1 receptor. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 4. pp. 2374-2385.
@article{0bb44fc0b55e4dea93ab2e94c1660af8,
title = "Identification of the hydrophobic ligand binding pocket of the S1P 1 receptor",
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.",
author = "Yuko Fujiwara and Osborne, {Daniel A.} and Walker, {Michelle D.} and Wang, {De An} and Bautista, {Debra A.} and Karoly Liliom and {Van Brocklyn}, {James R.} and Parrill, {Abby L.} and Gabor Tigyi",
year = "2007",
month = "1",
day = "26",
doi = "10.1074/jbc.M609648200",
language = "English (US)",
volume = "282",
pages = "2374--2385",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "4",

}

TY - JOUR

T1 - Identification of the hydrophobic ligand binding pocket of the S1P 1 receptor

AU - Fujiwara, Yuko

AU - Osborne, Daniel A.

AU - Walker, Michelle D.

AU - Wang, De An

AU - Bautista, Debra A.

AU - Liliom, Karoly

AU - Van Brocklyn, James R.

AU - Parrill, Abby L.

AU - Tigyi, Gabor

PY - 2007/1/26

Y1 - 2007/1/26

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=34047270109&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34047270109&partnerID=8YFLogxK

U2 - 10.1074/jbc.M609648200

DO - 10.1074/jbc.M609648200

M3 - Article

C2 - 17114791

AN - SCOPUS:34047270109

VL - 282

SP - 2374

EP - 2385

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 4

ER -