ABSTRACT
Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with at least six cognate G protein-coupled receptors. Herein, we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analyses. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.
Subject(s)
Crystallography, X-Ray , Binding Sites , Chromatography, Gel , Humans , Ligands , Models, Molecular , Receptors, Lysophosphatidic Acid/antagonists & inhibitors , Receptors, Lysosphingolipid/chemistry , Small Molecule LibrariesABSTRACT
ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans. Here we compare four x-ray structures of the bacterial ABC lipid flippase, MsbA, trapped in different conformations, two nucleotide-bound structures and two in the absence of nucleotide. Comparison of the nucleotide-free conformations of MsbA reveals a flexible hinge formed by extracellular loops 2 and 3. This hinge allows the nucleotide-binding domains to disassociate while the ATP-binding half sites remain facing each other. The binding of the nucleotide causes a packing rearrangement of the transmembrane helices and changes the accessibility of the transporter from cytoplasmic (inward) facing to extracellular (outward) facing. The inward and outward openings are mediated by two different sets of transmembrane helix interactions. Altogether, the conformational changes between these structures suggest that large ranges of motion may be required for substrate transport.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , Bacterial Proteins/chemistry , Escherichia coli Proteins/chemistry , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/metabolism , Adenylyl Imidodiphosphate/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Binding Sites , Biological Transport, Active , Crystallography, X-Ray , Escherichia coli Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Motion , Protein Binding , Protein Conformation , Salmonella typhimurium/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Vibrio cholerae/chemistryABSTRACT
The role of cholesterol in eukaryotic membrane protein function has been attributed primarily to an influence on membrane fluidity and curvature. We present the 2.8 A resolution crystal structure of a thermally stabilized human beta(2)-adrenergic receptor bound to cholesterol and the partial inverse agonist timolol. The receptors pack as monomers in an antiparallel association with two distinct cholesterol molecules bound per receptor, but not in the packing interface, thereby indicating a structurally relevant cholesterol-binding site between helices I, II, III, and IV. Thermal stability analysis using isothermal denaturation confirms that a cholesterol analog significantly enhances the stability of the receptor. A consensus motif is defined that predicts cholesterol binding for 44% of human class A receptors, suggesting that specific sterol binding is important to the structure and stability of other G protein-coupled receptors, and that this site may provide a target for therapeutic discovery.
Subject(s)
Cholesterol/chemistry , Receptors, Adrenergic, beta-2/chemistry , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Protein Structure, Secondary , Temperature , Timolol/chemistryABSTRACT
The 2.4 A crystal structure of the beta(2)-adrenergic receptor (beta(2)AR) in complex with the high-affinity inverse agonist (-)-carazolol provides a detailed structural framework for the analysis of ligand recognition by adrenergic receptors. Insights into agonist binding and the corresponding conformational changes triggering G-protein coupled receptor (GPCR) activation mechanism are of special interest. Here we show that while the carazolol pocket captured in the beta(2)AR crystal structure accommodates (-)-isoproterenol and other agonists without steric clashes, a finite movement of the flexible extracellular part of TM-V helix (TM-Ve) obtained by receptor optimization in the presence of docked ligand can further improve the calculated binding affinities for agonist compounds. Tilting of TM-Ve towards the receptor axis provides a more complete description of polar receptor-ligand interactions for full and partial agonists, by enabling optimal engagement of agonists with two experimentally identified anchor sites, formed by Asp113/Asn312 and Ser203/Ser204/Ser207 side chains. Further, receptor models incorporating a flexible TM-V backbone allow reliable prediction of binding affinities for a set of diverse ligands, suggesting potential utility of this approach to design of effective and subtype-specific agonists for adrenergic receptors. Systematic differences in capacity of partial, full and inverse agonists to induce TM-V helix tilt in the beta(2)AR model suggest potential role of TM-V as a conformational "rheostat" involved in the whole spectrum of beta(2)AR responses to small molecule signals.
Subject(s)
Adrenergic beta-2 Receptor Agonists , Drug Partial Agonism , Isoproterenol/pharmacology , Propanolamines/pharmacology , Receptors, Adrenergic, beta-2/chemistry , Adrenergic beta-2 Receptor Antagonists , Albuterol/chemistry , Albuterol/pharmacology , Amphetamines/chemistry , Amphetamines/pharmacology , Binding Sites , Catechols/chemistry , Catechols/pharmacology , Dopamine/chemistry , Dopamine/pharmacology , Hydroxyquinolines/chemistry , Hydroxyquinolines/pharmacology , Isoproterenol/chemistry , Kinetics , Ligands , Models, Molecular , Pliability/drug effects , Propanolamines/chemistry , Protein Structure, Secondary , Structure-Activity Relationship , ThermodynamicsABSTRACT
Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of a fusion partner that was inserted into the third intracellular loop. Using chimeras of the human ß(2)-adrenergic and human A(2A) adenosine receptors, we present the methodology and data for the initial selection of an expanded set of fusion partners for crystallizing GPCRs. In particular, use of the thermostabilized apocytochrome b(562)RIL as a fusion partner displays certain advantages over previously utilized fusion proteins, resulting in a significant improvement in stability and structure of GPCR-fusion constructs.
Subject(s)
Cytochromes b/chemistry , Muramidase/chemistry , Receptor, Adenosine A2A/chemistry , Recombinant Fusion Proteins/chemistry , Amino Acid Sequence , Animals , Cell Line , Chromatography, Gel , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Cytochromes b/biosynthesis , Cytochromes b/isolation & purification , Humans , Molecular Sequence Data , Muramidase/biosynthesis , Muramidase/isolation & purification , Protein Stability , Receptor, Adenosine A2A/biosynthesis , Receptor, Adenosine A2A/isolation & purification , Receptors, G-Protein-Coupled/biosynthesis , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purificationABSTRACT
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.
Subject(s)
Receptors, Lysosphingolipid/chemistry , Anilides/chemistry , Binding Sites , Crystallography, X-Ray , Models, Molecular , Muramidase/chemistry , Mutagenesis , Organophosphonates/chemistry , Protein Conformation , Receptors, Lysosphingolipid/agonists , Receptors, Lysosphingolipid/antagonists & inhibitors , Receptors, Lysosphingolipid/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/geneticsABSTRACT
Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A(2A) adenosine receptor by replacing its third intracellular loop with apocytochrome b(562)RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp(2.50). Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.
Subject(s)
Receptor, Adenosine A2A/chemistry , Receptor, Adenosine A2A/metabolism , Sodium/analysis , Adenosine A2 Receptor Agonists/metabolism , Adenosine A2 Receptor Antagonists/metabolism , Allosteric Regulation , Cholesterol/chemistry , Crystallography, X-Ray , Cytochrome b Group/chemistry , Escherichia coli Proteins/chemistry , HEK293 Cells , Humans , Hydrogen Bonding , Ligands , Lipid Bilayers , Lipids/chemistry , Models, Molecular , Protein Conformation , Protein Engineering , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Triazines/metabolism , Triazoles/metabolism , Water/chemistryABSTRACT
G protein-coupled receptor (GPCR) instability represents one of the most profound obstacles in the structural study of GPCRs that bind diffusible ligands. The introduction of targeted mutations at nonconserved residues that lie proximal to helix interfaces has the potential to enhance the fold stability of the receptor helix bundle while maintaining wild-type receptor function. To test this hypothesis, we studied the effect of amino acid substitutions at Glu122(3.41) in the well-studied beta(2)-adrenergic receptor (beta(2)AR), which was predicted from sequence conservation to lie at a position equivalent to a tryptophan residue in rhodopsin at the 3,4,5 helix interface among transmembrane (TM) domains 3, 4, and 5. Replacement of Glu122(3.41) with bulky hydrophobic residues, such as tryptophan, tyrosine, and phenylalanine, increases the yield of functionally folded beta(2)AR by as much as 5-fold. Receptor stability in detergent solution was studied by isothermal denaturation, and it was found that the E122W and E122Y mutations enhanced the beta(2)AR thermal half-life by 9.3- and 6.7-fold, respectively, at 37 degrees C. The beta(1)AR was also stabilized by the introduction of tryptophan at Glu147(3.41), and the effect on protein behavior was similar to the rescue of the unstable wild-type receptor by the antagonist propranolol. Molecular modeling of the E122W and E122Y mutants revealed that the tryptophan ring edge and tyrosine hydroxyl are positioned proximal to the helical break in TM5 introduced by the conserved Pro211(5.50) and may stabilize the helix by interacting favorably with the unpaired carbonyl oxygen of Val206(5.45). Conformational flexibility of TM5 is likely to be a general property of class A GPCRs; therefore, engineering of the TM4-TM3-TM5 interface at the 3.41 position may provide a general strategy for the stabilization of other receptors.