Michael A. Hanson

High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor

Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human β2-adrenergic receptor–T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein–coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the β2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.

The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist

The adenosine class of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.

The 2.6 Angstrom Crystal Structure of a Human A[subscript 2A] Adenosine Receptor Bound to an Antagonist

The adenosine class of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.

GPCR Engineering Yields High-Resolution Structural Insights into β2-Adrenergic Receptor Function

The β2-adrenergic receptor (β2AR) is a well-studied prototype for heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the β2AR and to facilitate its crystallization, we engineered a β2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR (“β2AR-T4L”) and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of β2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.

Structure of the human dopamine d3 receptor in complex with a d2/d3 selective antagonist.

Tweaking Dopamine Reception Dopamine modulates many cognitive and emotional functions of the human brain by activating G protein–coupled receptors. Antipsychotic drugs that block two of the receptor subtypes are used to treat schizophrenia but have multiple side effects. Chien et al. (p. 1091; see the Research Article by Wu et al.) resolved the crystal structure of one receptor in complex with a small-molecule inhibitor at 3.15 angstrom resolution. Homology modeling with other receptor subtypes might be a promising route to reveal potential structural differences that can be exploited in the design of selective therapeutic inhibitors having fewer side effects. Discovery of a binding site in the extracellular domain of a dopamine receptor offers hope for more selective therapeutics. Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein–coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.

Crystal structure of a lipid G protein-coupled receptor.

A Lipid-Sensing GPCR Sphingosine 1-phosphate (S1P) is a sphingolipid that binds to the G protein–coupled receptor subtype 1 (S1P1) to activate signaling pathways involved in regulation of the vascular and immune systems. Hanson et al. (p. 851) determined the crystal structure of S1PR in complex with an antagonist sphingolipid mimic. Ligand access to the receptor from the extracellular milieu is occluded, and a gap between helices I and VII may provide ligand access from within the membrane. The structural information, together with mutagenesis and structure activity relationship data, provides insight into the molecular recognition events that modulate signaling. A channel in a lipid-dependent G protein–coupled receptor allows a ligand to access its binding site from within the plasma membrane. 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 (S1P1-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 S1P1, resulting in the modulation of immune and stromal cell responses.

Microscale Fluorescent Thermal Stability Assay for Membrane Proteins

Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).

Discovery of new GPCR biology: one receptor structure at a time.

G-protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome. Within the last year, we have witnessed a relative explosion in the amount of structural information available for the GPCR family with two new structures of opsin in the presence and absence of transducin peptide, four new structures of beta-adrenergic receptors, and a recent structure of the human adenosine A2A receptor. The new biological insight being gained, such as the highly divergent extracellular loops and areas of structural convergence within the transmembrane helices, allows us to chart a course for further investigation into this important class of membrane proteins.

Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 A resolution.

Botulinum neurotoxin serotype B is a zinc protease that disrupts neurotransmitter release by cleaving synaptobrevin-II (Sb2), one of three SNARE proteins involved in neuronal synaptic vesicle fusion. The three-dimensional crystal structure of the apo botulinum neurotoxin serotype B catalytic domain (BoNT/B-LC) has been determined to 2.2 Å resolution, and the complex of cleaved Sb2 with the catalytic domain (Sb2–BoNT/B-LC) has been determined to 2.0 Å resolution. A comparison of the holotoxin catalytic domain and the isolated BoNT/B-LC structure shows a rearrangement of three active site loops. This rearrangement exposes the BoNT/B active site. The Sb2–BoNT/B-LC structure illustrates two distinct binding regions, which explains the specificity of each botulinum neurotoxin for its synaptic vesicle protein. This observation provides an explanation for the proposed cooperativity between binding of full-length substrate and catalysis and suggest a mechanism of synaptobrevin proteolysis employed by the clostridial neurotoxins.

Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam

Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 µm minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets.