5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology.

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.

Crystal Structure of the Human Cannabinoid Receptor CB1.

Cannabinoid receptor 1 (CB1) is the principal target of Δ9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

Crystal Structure of Antagonist Bound Human Lysophosphatidic Acid Receptor 1.

Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with at least six cognate G protein-coupled receptors. Herein, we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analyses. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.

Sphingosine-1-phosphate and its receptors: structure, signaling, and influence.

The sphingosine-1-phosphate (S1P) receptor signaling system has biological and medical importance and is the first lipid G protein-coupled receptor (GPCR) structure to be solved to 2.8-Å resolution. S1P binds to five high-affinity GPCRs generating multiple downstream signals that play essential roles in vascular development and endothelial integrity, control of cardiac rhythm, and routine oral treatment of multiple sclerosis. Genetics, chemistry, and now structural biology have advanced this integrated biochemical system. The S1P receptors have a novel N-terminal fold that occludes access to the binding pocket from the extracellular environment as well as orthosteric and bitopic ligands with very different physicochemical properties. S1P receptors and metabolizing enzymes have been deleted, inducibly deleted, and knocked in as tagged or altered receptors in mice. An array of genetic models allows analysis of integrated receptor function in vivo. We can now directly understand causal relationships among protein expression, signal, and control points in physiology and pathology.

The developing bird pelvis passes through ancestral dinosaurian conditions.

Living birds (Aves) have bodies substantially modified from the ancestral reptilian condition. The avian pelvis in particular experienced major changes during the transition from early archosaurs to living birds1,2. This stepwise transformation is well documented by an excellent fossil record2-4; however, the ontogenetic alterations that underly it are less well understood. We used embryological imaging techniques to examine the morphogenesis of avian pelvic tissues in three dimensions, allowing direct comparison with the fossil record. Many ancestral dinosaurian features2 (for example, a forward-facing pubis, short ilium and pubic 'boot') are transiently present in the early morphogenesis of birds and arrive at their typical 'avian' form after transitioning through a prenatal developmental sequence that mirrors the phylogenetic sequence of character acquisition. We demonstrate quantitatively that avian pelvic ontogeny parallels the non-avian dinosaur-to-bird transition and provide evidence for phenotypic covariance within the pelvis that is conserved across Archosauria. The presence of ancestral states in avian embryos may stem from this conserved covariant relationship. In sum, our data provide evidence that the avian pelvis, whose early development has been little studied5-7, evolved through terminal addition-a mechanism8-10 whereby new apomorphic states are added to the end of a developmental sequence, resulting in expression8,11 of ancestral character states earlier in that sequence. The phenotypic integration we detected suggests a previously unrecognized mechanism for terminal addition and hints that retention of ancestral states in development is common during evolutionary transitions.

Complete Ichthyornis skull illuminates mosaic assembly of the avian head.

The skull of living birds is greatly modified from the condition found in their dinosaurian antecedents. Bird skulls have an enlarged, toothless premaxillary beak and an intricate kinetic system that includes a mobile palate and jaw suspensorium. The expanded avian neurocranium protects an enlarged brain and is flanked by reduced jaw adductor muscles. However, the order of appearance of these features and the nature of their earliest manifestations remain unknown. The Late Cretaceous toothed bird Ichthyornis dispar sits in a pivotal phylogenetic position outside living groups: it is close to the extant avian radiation but retains numerous ancestral characters1-3. Although its evolutionary importance continues to be affirmed3-8, no substantial new cranial material of I. dispar has been described beyond incomplete remains recovered in the 1870s. Jurassic and Cretaceous Lagerstätten have yielded important avialan fossils, but their skulls are typically crushed and distorted 9 . Here we report four three-dimensionally preserved specimens of I. dispar-including an unusually complete skull-as well as two previously overlooked elements from the Yale Peabody Museum holotype, YPM 1450. We used these specimens to generate a nearly complete three-dimensional reconstruction of the I. dispar skull using high-resolution computed tomography. Our study reveals that I. dispar had a transitional beak-small, lacking a palatal shelf and restricted to the tips of the jaws-coupled with a kinetic system similar to that of living birds. The feeding apparatus of extant birds therefore evolved earlier than previously thought and its components were functionally and developmentally coordinated. The brain was relatively modern, but the temporal region was unexpectedly dinosaurian: it retained a large adductor chamber bounded dorsally by substantial bony remnants of the ancestral reptilian upper temporal fenestra. This combination of features documents that important attributes of the avian brain and palate evolved before the reduction of jaw musculature and the full transformation of the beak.

Structure of the glucagon receptor in complex with a glucagon analogue.

Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation-which requires conformational changes of the stalk, first extracellular loop and TMD-that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.

Decelerated dinosaur skull evolution with the origin of birds.

The evolutionary radiation of birds has produced incredible morphological variation, including a huge range of skull form and function. Investigating how this variation arose with respect to non-avian dinosaurs is key to understanding how birds achieved their remarkable success after the Cretaceous-Paleogene extinction event. Using a high-dimensional geometric morphometric approach, we quantified the shape of the skull in unprecedented detail across 354 extant and 37 extinct avian and non-avian dinosaurs. Comparative analyses reveal fundamental differences in how skull shape evolved in birds and non-avian dinosaurs. We find that the overall skull shape evolved faster in non-avian dinosaurs than in birds across all regions of the cranium. In birds, the anterior rostrum is the most rapidly evolving skull region, whereas more posterior regions-such as the parietal, squamosal, and quadrate-exhibited high rates in non-avian dinosaurs. These fast-evolving elements in dinosaurs are strongly associated with feeding biomechanics, forming the jaw joint and supporting the jaw adductor muscles. Rapid pulses of skull evolution coincide with changes to food acquisition strategies and diets, as well as the proliferation of bony skull ornaments. In contrast to the appendicular skeleton, which has been shown to evolve more rapidly in birds, avian cranial morphology is characterised by a striking deceleration in morphological evolution relative to non-avian dinosaurs. These results may be due to the reorganisation of skull structure in birds-including loss of a separate postorbital bone in adults and the emergence of new trade-offs with development and neurosensory demands. Taken together, the remarkable cranial shape diversity in birds was not a product of accelerated evolution from their non-avian relatives, despite their frequent portrayal as an icon of adaptive radiations.

Crystal structures of agonist-bound human cannabinoid receptor CB1.

The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC). Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1-agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a 'twin toggle switch' of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros-Weinstein numbering) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties.

Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators.

The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis. The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner. Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR and located outside helices V-VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs. Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling.

Structure of the full-length glucagon class B G-protein-coupled receptor.

The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a ß-strand conformation, instead of forming an α-helix as observed in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a ß-hairpin conformation and interacts with the stalk to form a compact ß-sheet structure. Hydrogen-deuterium exchange, disulfide crosslinking and molecular dynamics studies suggest that the stalk and the first extracellular loop have critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors.

An online resource for GPCR structure determination and analysis.

G-protein-coupled receptors (GPCRs) transduce physiological and sensory stimuli into appropriate cellular responses and mediate the actions of one-third of drugs. GPCR structural studies have revealed the general bases of receptor activation, signaling, drug action and allosteric modulation, but so far cover only 13% of nonolfactory receptors. We broadly surveyed the receptor modifications/engineering and methods used to produce all available GPCR crystal and cryo-electron microscopy (cryo-EM) structures, and present an interactive resource integrated in GPCRdb ( http://www.gpcrdb.org ) to assist users in designing constructs and browsing appropriate experimental conditions for structure studies.

Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor.

Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging owing to its affordability, stability, ease of administration and clinical efficacy. However, misoprostol lacks receptor and tissue selectivities, and thus its use is accompanied by a number of serious side effects. The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction. Here, we present the 2.5 Å resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E2 receptor 3 (EP3). The active state structure reveals a completely enclosed binding pocket containing a structured water molecule that coordinates misoprostol's ring structure. Modeling of selective agonists in the EP3 structure reveals rationales for selectivity. These findings will provide the basis for the next generation of uterotonic drugs that will be suitable for administration in low resource settings.

Publisher Correction: Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor.

In the version of this article originally published, the present address for Petr Popov was incorrectly listed as 'Koltech Institute of Science & Technology, Moscow, Russia'. The correct present address is 'Skolkovo Institute of Science and Technology, Moscow, Russia'. The error has been corrected in the HTML and PDF versions of the paper.

Structural Determinants of Binding the Seven-transmembrane Domain of the Glucagon-like Peptide-1 Receptor (GLP-1R).

The glucagon-like peptide-1 receptor (GLP-1R) belongs to the secretin-like (class B) family of G protein-coupled receptors. Members of the class B family are distinguished by their large extracellular domain, which works cooperatively with the canonical seven-transmembrane (7TM) helical domain to signal in response to binding of various peptide hormones. We have combined structure-based site-specific mutational studies with molecular dynamics simulations of a full-length model of GLP-1R bound to multiple peptide ligand variants. Despite the high sequence similarity between GLP-1R and its closest structural homologue, the glucagon receptor (GCGR), nearly half of the 62 stably expressed mutants affected GLP-1R in a different manner than the corresponding mutants in GCGR. The molecular dynamics simulations of wild-type and mutant GLP-1R·ligand complexes provided molecular insights into GLP-1R-specific recognition mechanisms for the N terminus of GLP-1 by residues in the 7TM pocket and explained how glucagon-mimicking GLP-1 mutants restored binding affinity for (GCGR-mimicking) GLP-1R mutants. Structural analysis of the simulations suggested that peptide ligand binding mode variations in the 7TM binding pocket are facilitated by movement of the extracellular domain relative to the 7TM bundle. These differences in binding modes may account for the pharmacological differences between GLP-1 peptide variants.

Structural biology of the S1P1 receptor.

The sphingosine 1 phosphate receptor family has been studied widely since the initial discovery of its first member, endothelium differentiation gene 1. Since this initial discovery, the family has been renamed and the primary member of the family, the S1P1 receptor, has been targeted for a variety of disease indications and successfully drugged for the treatment of patients with relapsing multiple sclerosis. Recently, the three-dimensional structure of the S1P1 receptor has been determined by X-ray crystallography and the specifics of the sphingosine 1 phosphate ligand binding pocket mapped. Key structural features for the S1P1 receptor will be reviewed and the potential binding modes of additional pharmacologically active agents against the receptor will be analyzed in an effort to better understand the structural basis of important receptor-ligand interactions.

G protein ß4 as a structural determinant of enhanced nucleotide exchange in the A2AAR-Gs complex.

Adenosine A2A receptors (A2AAR) evoke pleiotropic intracellular signaling events via activation of the stimulatory heterotrimeric G protein, Gs. Here, we used cryoEM to solve the agonist-bound structure of A2AAR in a complex with full-length Gs α and Gß4γ2 (A2AAR-Gs α:ß4γ2). The orthosteric binding site of A2AAR-Gs α:ß4γ2 was similar to other structures of agonist-bound A2AAR, with or without Gs. Unexpectedly, the solvent accessible surface area within the interior of the complex was substantially larger for the complex with Gß4 versus the closest analog, A2AAR-miniGs α:ß1γ2. Consequently, there are fewer interactions between the switch II in Gs α and the Gß4 torus. In reconstitution experiments Gß4γ2 displayed a ten-fold higher efficiency over Gß1γ2 in catalyzing A2AAR dependent GTPγS binding to Gs α. We propose that the less constrained switch II in A2AAR-Gs α:ß4γ2 accounts for this increased efficiency. These results suggest that Gß4 functions as a positive allosteric enhancer versus Gß1.

High pollutant removal efficacy of a large constructed wetland leads to receiving stream improvements.

Hewletts Creek, in Wilmington, North Carolina, drains a large suburban watershed and as such is affected by high fecal bacteria loads and periodic algal blooms from nutrient loading. During 2007, a 3.1-ha wetland was constructed to treat stormwater runoff from a 238-ha watershed within the Hewletts Creek drainage. A rain event sampling program was performed in 2009-2010 to evaluate the efficacy of the wetland in reducing pollutant loads from the stormwater runoff passing through the wetland. During the eight storms sampled, the wetland greatly moderated the hydrograph and retained and/or removed 50 to 75% of the inflowing stormwater volume. High removal rates of fecal coliform bacteria were achieved, with an average load reduction of 99% and overall concentration reduction of >90%. Particularly high (>90%) reductions of ammonium and orthophosphate loads also occurred, and lesser but still substantial reductions of total phosphorus (89%) and total suspended solids loads (88%) were achieved. Removal of nitrate was seasonally dependent, with lower removal occurring in cold weather and a high percentage (90%+) of nitrate load removal occurring in the growing season when water temperature exceeded 15°C. Long-term before-and-after sampling in downstream Hewletts Creek proper showed that, after wetland construction, statistically significant average decreases of 43% for nitrate, 72% for ammonium, and 59% for fecal coliform bacteria were realized. Wetland features contributing to the high pollutant control efficacy included available space for a large wetland, construction of deep forebays, and a dense and diverse aquatic and shoreline plant assemblage.

Response to Comment on "The early origin of a birdlike inner ear and the evolution of dinosaurian movement and vocalization".

David et al. claim that vestibular shape does not reflect function and that we did not use phylogenetic inference methods in our primary analyses. We show that their claims are countered by comparative and direct experimental evidence from across Vertebrata and that their models are empirically unverified. We did use phylogenetic methods to test our hypotheses. Moreover, their phylogenetic correction attempts are methodologically inappropriate.