The adenosine class of G protein-coupled receptors 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 A 2A adenosine receptor in complex with a high affinity subtypeselective antagonist, ZM241385, to 2.6 Å 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 defines 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 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.

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. Access to the binding pocket is completely occluded by the N-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 hydrophobic volume triggering that activates S1P1 resulting in the modulation of immune and stromal cell responses.

SUMMARY Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of the T4 lysozyme fusion partner that was inserted into the third intracellular loop. Using chimeras of the human β2-adrenergic and human A2A adenosine receptors, we present the methodology and data for the selection of five new fusion partners for crystallizing GPCRs. In particular, the use of the thermostabilized apocytochrome b562RIL as a fusion partner displays certain advantages over the previously utilized T4 lysozyme, resulting in a significant improvement in stability and structure in GPCR-fusion constructs.

The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 A resolution. The structure of this "X" domain, seen in many single-stranded RNA viruses, reveals a three-layered alpha/beta/alpha core with a macro-H2A-like fold. The putative active site is a solvent-exposed cleft that is conserved in its three structural homologs, yeast Ymx7, Archeoglobus fulgidus AF1521, and Er58 from E. coli. Its sequence is similar to yeast YBR022W (also known as Poa1P), a known phosphatase that acts on ADP-ribose-1''-phosphate (Appr-1''-p). The SARS nsP3 domain readily removes the 1'' phosphate group from Appr-1''-p in in vitro assays, confirming its phosphatase activity. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1''-p.