muramidase and carazolol

Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor-receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the β2-adrenergic receptor (β2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For β2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a β2-AR/T4-lysozyme chimera bound to the antagonist carazolol.

Topics: Ammonium Sulfate; Animals; Cattle; Centrifugation, Density Gradient; Chromatography, Affinity; Chromatography, Gel; Crystallization; Crystallography, X-Ray; Glucosides; Muramidase; Propanolamines; Protein Multimerization; Protein Structure, Secondary; Receptors, Adrenergic, beta-2; Recombinant Fusion Proteins; Rhodopsin; Rod Cell Outer Segment; Sf9 Cells; Spodoptera; Viral Proteins; Zinc Acetate

The beta2-adrenergic receptor (beta2AR) 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 beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-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 beta2AR-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.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Amino Acid Sequence; Bacteriophage T4; Binding Sites; Cell Line; Cell Membrane; Crystallization; Crystallography, X-Ray; Drug Inverse Agonism; Humans; Immunoglobulin Fab Fragments; Ligands; Models, Molecular; Molecular Sequence Data; Muramidase; Propanolamines; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Adrenergic, beta-2; Recombinant Fusion Proteins

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 beta2-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 beta2-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.

Topics: Bacteriophage T4; Binding Sites; Cell Membrane; Cholesterol; Crystallization; Crystallography, X-Ray; Drug Inverse Agonism; Humans; Ligands; Models, Molecular; Muramidase; Propanolamines; Protein Conformation; Protein Folding; Protein Structure, Secondary; Receptors, Adrenergic, beta-2; Recombinant Fusion Proteins; Rhodopsin; Static Electricity

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Bacteriophage T4; Binding Sites; Cell Membrane; Immunoglobulin Fab Fragments; Ligands; Muramidase; Propanolamines; Protein Conformation; Protein Folding; Protein Structure, Secondary; Receptors, Adrenergic, beta-2; Recombinant Fusion Proteins; Rhodopsin; Signal Transduction