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Activation of G protein-coupled receptors upon agonist binding is a critical step in the signaling cascade for this family of cell surface proteins. We report the crystal structure of the A(2A) adenosine receptor (A(2A)AR) bound to an agonist UK-432097 at 2.7 angstrom resolution. Relative to inactive, antagonist-bound A(2A)AR, the agonist-bound structure displays an outward tilt and rotation of the cytoplasmic half of helix VI, a movement of helix V, and an axial shift of helix III, resembling the changes associated with the active-state opsin structure. Additionally, a seesaw movement of helix VII and a shift of extracellular loop 3 are likely specific to A(2A)AR and its ligand. The results define the molecule UK-432097 as a \"conformationally selective agonist\" capable of receptor stabilization in a specific active-state configuration.

Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

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 stremal cell responses.

Structural data show that the light atom at the center of the nitrogenase active site cofactor is a carbon. The identity of the interstitial light atom in the center of the FeMo cofactor of nitrogenase has been enigmatic since its discovery. Atomic-resolution x-ray diffraction data and an electron spin echo envelope modulation (ESEEM) analysis now provide direct evidence that the ligand is a carbon species.

Adenosine A 2A receptor structure Adenosine receptors are G protein-coupled receptors that are found in the heart and the brain, and adenosine is the endogenous ligand for this class of transmembrane receptor. Lebon et al . present two X-ray crystal structures of a thermostabilized human adenosine A 2A receptor bound to its endogenous agonist adenosine and the synthetic agonist NECA. Comparison of the agonist-bound structures of A 2A receptor with the agonist-bound structures of β-adrenoceptors suggests that the contraction of the ligand binding pocket caused by the inward motion of several helices may be a common feature in the activation of all G protein-coupled receptors. Adenosine receptors and β-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine 1 or noradrenaline 2 , respectively. GPCRs have similar structures consisting of seven transmembrane helices that contain well-conserved sequence motifs, indicating that they are probably activated by a common mechanism 3 , 4 . Recent structures of β-adrenoceptors highlight residues in transmembrane region 5 that initially bind specifically to agonists rather than to antagonists, indicating that these residues have an important role in agonist-induced activation of receptors 5 , 6 , 7 . Here we present two crystal structures of the thermostabilized human adenosine A 2A receptor (A 2A R-GL31) bound to its endogenous agonist adenosine and the synthetic agonist NECA. The structures represent an intermediate conformation between the inactive and active states, because they share all the features of GPCRs that are thought to be in a fully activated state, except that the cytoplasmic end of transmembrane helix 6 partially occludes the G-protein-binding site. The adenine substituent of the agonists binds in a similar fashion to the chemically related region of the inverse agonist ZM241385 (ref. 8 ). Both agonists contain a ribose group, not found in ZM241385, which extends deep into the ligand-binding pocket where it makes polar interactions with conserved residues in H7 (Ser 277 7.42 and His 278 7.43 ; superscripts refer to Ballesteros–Weinstein numbering 9 ) and non-polar interactions with residues in H3. In contrast, the inverse agonist ZM241385 does not interact with any of these residues and comparison with the agonist-bound structures indicates that ZM241385 sterically prevents the conformational change in H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures of A 2A R with the agonist-bound structures of β-adrenoceptors indicates that the contraction of the ligand-binding pocket caused by the inward motion of helices 3, 5 and 7 may be a common feature in the activation of all GPCRs.

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 Å resolution crystal structure of a β 1 -adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey ( Meleagris gallopavo ) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the β 1 -adrenergic receptor and binding of carazolol to the β 2 -adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β 2 -adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation. G-protein-coupled receptors: Binding commitment The adrenalin stress hormone receptor (β 1 adrenergic receptor or β 1 AR) regulates heart rate and blood pressure and is the target for β-blockers. Like other members of the G-protein-coupled receptor family, it is difficult to purify. But the form of the enzyme found in the turkey is more stable than the human equivalent, and by using that, and mutagenesis to thermostabilize the receptor, β 1 AR has been crystallized bound to the β-blocker cyano-pindolol. The structure reveals insights into the G-protein-binding interface.

The plasma membrane protein Orai forms the pore of the calcium release—activated calcium (CRAC) channel and generates sustained cytosolic calcium signals when triggered by depletion of calcium from the endoplasmic reticulum. The crystal structure of Orai from Drosophila melanogaster, determined at 3.35 angstrom resolution, reveals that the calcium channel is composed of a hexameric assembly of Orai subunits arranged around a central ion pore. The pore traverses the membrane and extends into the cytosol. A ring of glutamate residues on its extracellular side forms the selectivity filter. A basic region near the intracellular side can bind anions that may stabilize the closed state. The architecture of the channel differs markedly from other ion channels and gives insight into the principles of selective calcium permeation and gating.

Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human β 2 adrenoceptor (β 2 AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 Å/3.7 Å resolution. The cytoplasmic ends of the β 2 AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the β 2 AR are not seen. The β 2 AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the β 2 AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs. A tough structure to crack Most hormones and neurotransmitters — and therefore many drugs — work via G protein-coupled receptors, or GPCRs. With the one exception of rhodopsin, the most stable GPCR known, structural data for these proteins are hard to come by. Now several collaborating research groups, publishing in this issue of Nature and also in Science , have exploited a raft of different techniques, including the use of the inverse agonist carazolol to stabilize the receptor structure, to determine the crystal structure of the human β 2 AR adrenaline receptor. Its structure contrasts markedly with that of 'dark' rhodopsin, which helps explain why it is so hard to prepare diffraction-quality crystals of most GPCRs. High resolution structural information for G protein-coupled receptors has so far been limited to rhodopsin; here a crystal structure of the β 2 AR adrenaline receptor is presented.

P2X receptors are trimeric ATP-activated ion channels permeable to Na + , K + and Ca 2+ . The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X 4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body β-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents. The X-ray crystal structure of the zebrafish P2X 4 receptor in the presence and absence of ATP is determined, revealing an ATP-binding site and an open ion channel pore. ATP-gated P2X receptor structures P2X receptors are ATP-activated ion channels that are permeable to Na + , K + and Ca 2+ ions. These proteins are involved in a broad range of physiological processes, including the modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. In this study, the authors report X-ray crystal structures of the zebrafish P2X 4 receptor in the presence and absence of ATP. The ATP-bound structure reveals a previously unseen ATP-binding motif, and comparison of the two structures indicates that ATP binding leads to a radial expansion of the extracellular vestibule, which causes an iris-like expansion of the transmembrane helices, opening the ion channel.

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 b⁵⁶² 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.