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G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography (1-3), the details of ligand-induced [...]

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons1. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders (2). BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport (1-3). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals (4). Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other (5): gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin (5). Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor (6). In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain (7), but themolecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open f-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties., The overall structure of BoNT/A-RBD in complex with SV2C-LD is shown in Fig. 1, and the data collection and refinement statistics are listed in Extended Data Table 1. The convex [...]

G-protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters and sensory stimuli. Although some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state (1,2). However, these structures are for the apoprotein, or opsin, form that does not contain the agonist all-trans retinal. Here we present a crystal structure at a resolution of 3 Å for the constitutively active rhodopsin mutant Glu 113 Gln (3-5) in complex with a peptide derived from the carboxy terminus of the a-subunit of the G protein transducin. The protein is in an active conformation that retains retinal in the binding pocket after photoactivation. Comparison with the structure of ground-state rhodopsin (6) suggests how translocation of the retinal β-ionone ring leads to a rotation of transmembrane helix 6, which is the critical conformational change on activation (7). A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. We thus show how an agonist ligand can activate its GPCR., In the dark, rhodopsin contains a covalently bound 11-cis retinal chromophore that preferentially binds to the inactive state and therefore functions as an inverse agonist in the visual system. On [...]

β-adrenergic receptors (βARs) are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline (1, 2). Synthetic ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilized turkey (Meleagris gallopavo) [β.sub.1]-adrenergic receptor ([β.sub.1]AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine-binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 ([Ser.sup.5.42] and [Ser.sup.5.46]), but partial agonists only interact with [Ser.sup.5.42] (superscripts refer to Ballesteros-Weinstein numbering (3)). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies., Determining how agonists and antagonists bind to the b receptors has been the goal of research for more than 20 years (4-11). Although the structures of the homologous [β.sup.1] and [...]

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision (1,2). Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation (3,4). However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-L-phenylalanine residues incorporated at selected sites (5), and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II (3). Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures (6,7) and to understand conformational sub-states observed during the activation of other GPCRs (8)., GPCRs mediate transmembrane signal transduction by activating cytoplasmic G proteins, which in turn modulate the activity of downstream effector proteins. Many current models for GPCR activation are derived from the [...]

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7Å resolution crystal structure of a [beta.sub.1]-adrenergic receptor [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tips.2007.11.009 Byline: Brian Kobilka (1), Gebhard F.X. Schertler (2) Abstract: G-protein-coupled receptors (GPCRs) constitute a large family of structurally similar proteins that respond to a chemically diverse array of physiological and environmental stimulants. Until recently, high-resolution structural information was limited to rhodopsin, a naturally abundant GPCR that is highly specialized for the detection of light. Non-rhodopsin GPCRs for diffusible hormones and neurotransmitters have proven more resistant to crystallography approaches, possibly because of their inherent structural flexibility and the need for recombinant expression. Recently, crystal structures of the human [beta].sub.2 adrenoceptor have been obtained using two different approaches to stabilize receptor protein and increase polar surface area. These structures, together with the existing structures of rhodopsin, represent an important first step in understanding how GPCRs work at a molecular level. Much more high-resolution information is needed for this important family of membrane proteins, however: for example, the structures of GPCRs from different families, structures bound to ligands having different efficacies, and structures of GPCRs in complex with G proteins and other signaling molecules. Methods to characterize the dynamic aspects of the GPCR architecture at high resolution will also be important. Author Affiliation: (1) Departments of Molecular and Cellular Physiology and Medicine, Stanford University School of Medicine, 279 Campus Drive, Stanford, Palo Alto, CA 94305, USA (2) MRC Laboratory of Molecular Biology, Cambridge, CB2 0QT, UK

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 [...]

Bovine rhodopsin photointermediates formed in two-dimensional (2D) rhodopsin crystal suspensions are studied by measuring the time-dependent absorbance changes produced after excitation with 7 ns laser pulses at 15, 25, and 35 degree Celsius. A goal of the time-resolved absorbance measurements on suspensions of rhodopsin crystals is to determine the relationship of the photointermediates in those crystals to the normal bleaching sequence that appears after photoexcitation of more commonly studied preparations such as rhodopsin in membrane or detergent suspensions.

Research using a nine angstrom projection map of bovine rhodopsin shows that rhodopsin contains seven hydrophobic alpha-helices. The structure of frog rhodopsin has been established using electron micrographs of frozen-hydrated two-dimensional crystal samples. The tilt angles of each helix has been estimated. Evidence suggests that helices one and two are connected in some way.

Rhodopsin is the G protein-coupled receptor that upon light activation triggers the visual transduction cascade. Rod cell outer segment disc membranes were isolated from dark-adapted frog retinas and were extracted with Tween detergents to obtain two-dimensional rhodopsin crystals for electron crystallography. When Tween 80 was used, tubular structures with p2 lattice (a = 32 [Amstrong], b = 83 [Amstrong], [gamma] = 91 [degrees]) were formed. The use of a Tween 80/Tween 20 mixture favored the formation of larger [p22.sub.1][2.sub.1] lattices (a = 40 [Amstrong], b = 146 [Amstrong], [gamma] = 90 [degrees]). Micrographs from frozen hydrated frog rhodopsin crystals were processed, and projection structures to 7-[Amstrong] resolution for the [p22.sub.1][2.sub.1] form and to 6-[Amstrong] resolution for the p2 form were calculated. The maps of frog rhodopsin in both crystal forms are very similar to the 9-[Amstrong] map obtained previously for bovine rhodopsin and show that the arrangement of the helices is the same. In a tentative topographic model, helices 4, 6, and 7 are nearly perpendicular to the plane of the membrane. In the higher-resolution projection maps of frog rhodopsin, helix 5 looks more tilted than it appeared previously. The quality of the two frog rhodopsin crystals suggests that they would be suitable to obtain a three-dimensional structure in which all helices would be resolved.

A projection map of rhodopsin showing the configuration of its helices is presented. Rhodopsin is a visual pigment containing 348 amino acids, and has been characterized as having seveen helices crossing the disk membrane and an extracellular amino-terminus. Reconstitution of bovine rhodopsin into lipid bilayers yieelded 2-dimensional crystals, which were then analyzed by optical diffraction methods. The projection map provides a framework by which the abundant biochemical data for the protein can be interpreted.

Many invertebrates have excellent visual systems (1). Squid, for example, are formidable hunters that rely on their acute visual abilities to catch their prey. As in vertebrates, the properties of [...]