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. 2012 Feb 17;335(6070):851-5.
doi: 10.1126/science.1215904.

Crystal Structure of a Lipid G Protein-Coupled Receptor

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Free PMC article

Crystal Structure of a Lipid G Protein-Coupled Receptor

Michael A Hanson et al. Science. .
Free PMC article

Abstract

The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P(1)-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P(1), resulting in the modulation of immune and stromal cell responses.

Figures

Fig. 1
Fig. 1
Overview of the structural features unique to the S1P1 receptor. A. Ribbon trace of the receptor with relevant features highlighted. The receptor folds in a traditional seven transmembrane helical bundle similar to other class A GPCRs. However, the extracellular domain of the S1P1 receptor adopts a novel fold incorporating helical elements from the N-terminus (red ribbon), as well as ECL1 (gold ribbon), that occlude access to the ligand binding pocket. ECL2 (orange ribbon) and ECL3 (yellow ribbon) are both constrained by intraloop disulfide bonds (yellow ball-and-stick) and contribute important interactions within the binding pocket. The compound ML056 is shown in green carbon ball-and-stick rendering and W2696.48 is rendered as a space-filling atom in blue as reference point for the binding pocket. The T4L fusion protein was omitted from this figure for clarity. B. Top view of the receptor highlighting the critical role of the N-terminal helix in capping the ligand and limiting solvent access to the binding pocket. There is a sizable access point for ligand entry and exit between helices I and VII. C. Surface rendering of the receptor from the top view highlighting the occluded nature of the binding pocket with two points of access either for lipid or solvent. D. Side-view of the lipid access channel relative to the predicted position of the membrane bilayer.
Fig. 2
Fig. 2
Detailed structural representation of the interactions between ML056 and the S1P1 receptor. A. ML056 is colored as green carbons sticks. Polar residues within the binding pocket are colored with cyan carbons, whereas residues comprising the hydrophobic portion of the binding pocket are colored with tan carbons and the two disulfide bonds are shown in yellow ball-and-stick. The N-terminal helix and extracellular loops are shown as red and blue ribbons respectively. B. Two-dimensional residue interaction map illustrating the amino-acids within 4 Å of ML056. Polar interactions are represented by dotted cyan lines, whereas hydrophobic interactions are represented as solid yellow lines drawn to the closest atom of the ligand.
Fig. 3
Fig. 3
Docking study of ML056 analogs (green carbons) with progressively longer acyl chains. A ten carbon acyl chain (yellow carbons) switches the response from antagonism to agonism. A. Docking each of the five ligands into the antagonist binding pocket resulted in essentially superimposable docking poses for all of the ligands except ML056 + 4 (yellow carbons) which cannot maintain sphingolipid headgroup binding interactions due to high ligand strain. B. Docking each of the five ligands into the induced fit agonist modeled binding pocket reveals that all ligands can bind with reasonable docking scores while maintaining head group interactions. C. Table of docking and MM-GB/SA (Molecular Mechanics, Generalized Born and Solvent Accesibility) calculations for assessing the quality of the docking poses. The agonist compound ML056 + 4 docked into the antagonist binding pocket generates a steep drop in calculated ΔGbind along with an increase in calculated ligand strain.
Fig. 4
Fig. 4
A comparison of putative binding interactions for class I and class II S1P1 agonists. A. The docked binding pose of S1P in the modeled S1P1 receptor agonist binding pocket. The red and blue ribbons are from N-terminal and extracellular loops respectively. Residues that deviate from their crystallographically determined positions (tan carbons) are rendered twice, the red atoms represent the modeled agonist position determined by induced fit docking. B. The docked binding pose of CYM-5442 in the modeled agonist binding pocket. Residues that deviate from their crystallographically determined positions (tan carbons) are rendered twice, the red atoms represent the modeled agonist position determined by induced fit docking. The binding pocket must accommodate slightly greater ligand volume in the hydrophobic portion. C. Overlay of the docked ligands S1P (yellow carbons) and CYM-5442 (cyan carbons) compared to the structurally determined conformation of ML056 (green carbons). Binding pocket residues are indicated with black font for those that do not change between the agonist and antagonist binding models, red font for those that change for the agonist induced fit docking model, and green font for those that change conformation induced by CYM-5442 docking. D. Ligand-induced ERK phosphorylation in WT, W2696.48L, and W2696.48F Jump-In stable cell lines stimulated with increasing concentrations of either S1P or CYM-5442 (mean ± S.D. of triplicate samples). The data are from one of three independent experiments showing a minimal effect on S1P signaling. The progressive loss in potency of CYM-5442 tracks with a loss of aromatic π-stacking interactions. E. A differential loss of agonist responsiveness of the mutant W269L S1P1 receptor compared to WT receptor was observed. For each experiment, the EC50 for agonist activation of ERK phosphorylation of W269L S1P1 receptor was divided by the EC50 of WT S1P1 receptor.

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