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A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Salmeterol is a partial agonist for the β2 adrenergic receptor (β2AR), and the first long-acting β2AR agonist (LABA) to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol has been controversial both for its safety and mechanism of action. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound β2AR in complex with an active-state stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between β1AR and β2AR explain the high receptor subtype selectivity. A structural comparison with the β2AR bound to the full agonist epinephrine reveals differences in the hydrogen bond network involving residues Ser 2045.43 and Asn 2936.55. Mutagenesis and biophysical studies suggest that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G protein activation and the limited β-arrestin recruitment for salmeterol.

Salmeterol is a partial agonist for the β2 adrenergic receptor (β2AR) and the first long-acting β2AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol's safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound β2AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between β1AR and β2AR explain the high receptor-subtype selectivity. A structural comparison with the β2AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser2045.43 and Asn2936.55. Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited β-arrestin recruitment for salmeterol.

The recent increase in the number of X-ray crystal structures of G-protein coupled receptors (GPCRs) has been enabling for structure-based drug design (SBDD) efforts. These structures have revealed that GPCRs are highly dynamic macromolecules whose function is dependent on their intrinsic flexibility. Unfortunately, the use of static structures to understand ligand binding can potentially be misleading, especially in systems with an inherently high degree of conformational flexibility. Here, we show that docking a set of dopamine D3 receptor compounds into the existing eticlopride-bound dopamine D3 receptor (D3R) X-ray crystal structure resulted in poses that were not consistent with results obtained from site-directed mutagenesis experiments. We overcame the limitations of static docking by using large-scale high-throughput molecular dynamics (MD) simulations and Markov state models (MSMs) to determine an alternative pose consistent with the mutation data. The new pose maintains critical interactions observed in the D3R/eticlopride X-ray crystal structure and suggests that a cryptic pocket forms due to the shift of a highly conserved residue, F6.52. Our study highlights the importance of GPCR dynamics to understand ligand binding and provides new opportunities for drug discovery. N.F. acknowledges support from Generalitat de Catalunya (FI-Agaur). GDF acknowledges support from MINECO (BIO2014-53095-P) and FEDER. We also thank all the volunteers of GPUGRID who donated GPU computing time to the project.

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β(2)AR's ability to engage multiple signaling and regulatory proteins.

The enzyme phosphite dehydrogenase (PTDH) catalyzes the NAD+-dependent conversion of phosphite to phosphate and represents the first biological catalyst that has been characterized to carry out the enzymatic oxidation of phosphorus. Despite over a decade’s worth of investigation into both the mechanism of its unusual reaction, as well as its utility in cofactor regeneration, there has been a lack of any structural data on PTDH. Here we present the co-crystal structure of an engineered thermostable variant of PTDH bound to NAD+ (1.7 Å resolution), as well as four other co-crystal structures of thermostable PTDH and its variants with different ligands (all between 1.85 – 2.3 Å resolution). These structures provide a molecular framework for understanding prior mutational analysis, and point to additional residues, located in the active site, that may contribute to the enzymatic activity of this highly unusual catalyst.

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the β(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

In the version of this paper originally published, the structure for epinephrine shown in Figure 1a was redrawn with an extra carbon. The structure has been replaced in the HTML and PDF versions of the article. The original and corrected versions of the structure are shown below.

G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters, and these membrane proteins are the largest group of therapeutic targets for a broad range of diseases. It is very difficult to obtain high-resolution X-ray crystal structures of GPCRs; little is known about the functional role(s) of the extracellular surface in receptor activation or about the conformational coupling of the extracellular surface to the native ligand-binding pocket. In this study, Bokoch et al. used NMR spectroscopy to investigate ligand-specific conformational changes around a salt bridge linking extracellular loops 2 and 3 of the β2 adrenergic receptor. They found that drugs that bind within the transmembrane core (and exhibit different efficacies towards G-protein activation) stabilize distinct conformations of the extracellular surface. New therapeutic agents that target this diverse surface could function as allosteric modulators with high subtype selectivity. G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters and are the largest group of therapeutic targets for a range of diseases. The extracellular surface (ECS) of GPCRs is diverse and therefore an ideal target for the discovery of subtype-selective drugs. Here, NMR spectroscopy is used to investigate ligand-specific conformational changes around a central structural feature in the ECS of a GPCR. G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs1,2,3,4,5 have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

Summary The human pathogen Pseudomonas aeruginosa coordinates the expression of virulence factors using quorum sensing, a signaling cascade triggered by the activation of signal receptors by small-molecule autoinducers. These homoserine lactone autoinducers stabilize their cognate receptors and activate their functions as transcription factors. Because quorum sensing regulates the progression of infection and host immune resistance, significant efforts have been devoted toward the identification of small molecules that disrupt this process. Screening efforts have identified a class of triphenyl compounds that are structurally distinct from the homoserine lactone autoinducer, yet interact specifically and potently with LasR receptor to modulate quorum sensing (Muh et al., 2006a). Here we present the high-resolution crystal structures of the ligand binding domain of LasR in complex with the autoinducer N -3-oxo-dodecanoyl homoserine lactone (1.4 A resolution), and with the triphenyl mimics TP-1, TP-3, and TP-4 (to between 1.8 A and 2.3 A resolution). These crystal structures provide a molecular rationale for understanding how chemically distinct compounds can be accommodated by a highly selective receptor, and provide the framework for the development of novel quorum-sensing regulators, utilizing the triphenyl scaffold.

Recent crystal structures reveal the inactive states of non-rhodopsin G-protein coupled receptors (GPCRs) in beautiful detail. Solution NMR spectroscopy is ideally suited to contribute dynamic information regarding GPCR activation. However, these eukaryotically-expressed membrane proteins remain challenging NMR targets. We apply selective labeling with [13C]methyl probes and two-dimensional NMR to analyze ligand-induced conformational changes in beta2-adrenergic receptor (b2AR).Lysine side chains were labeled with [13C]dimethyl probes to explore conformational changes in the b2AR extracellular surface. Lys305 forms a salt bridge connecting the extracellular end of transmembrane (TM) helix 7 with extracellular loop 2. The Lys305 NMR resonances are sensitive to conformational changes in the receptor extracellular surface. Using NMR, we observe disruption of the Lys305 salt bridge upon receptor activation by agonist. Computational modeling suggests that a lateral displacement of TM7 occurs in concert with an inward motion at the extracellular end of TM6 (thus extending the “global toggle switch” model of Schwartz (2006) Annu. Rev. Pharmacol. Toxicol.) Different conformational changes occur upon inverse agonist binding. Molecular dynamics simulations suggest that a conserved phenylalanine (Phe193) in the orthosteric ligand binding site is key for inverse agonism. Taken as a whole, these results demonstrate conformational coupling between the GPCR extracellular surface and orthosteric ligand binding site within the transmembrane domains (Ahuja (2009) Nat. Struct. Mol. Biol.) This provides rationale for developing allosteric pharmaceuticals targeting the GPCR extracellular surface.Conformational changes within the b2AR transmembrane core are also observed by NMR using selective epsilon-[13CH3] labeling of methionines. While assignments are pending, clear conformational changes are seen with activation or inverse agonist binding. [13C]methyl NMR spectroscopy, in combination with crystal structures and molecular dynamics simulation, provides a dynamic view of the conformational changes intrinsic to GPCR function.

SummaryThe lipoglycopeptide antibiotics teicoplanin and A40926 have proven efficacy against Gram-positive pathogens. These drugs are distinguished from glycopeptide antibiotics by N-linked long chain acyl-D-glucosamine decorations that contribute to antibacterial efficacy. During the biosynthesis of lipoglycopeptides, tailoring glycosyltransferases attach an N-acetyl-D-glucosamine to the aglycone, and this N-acetyl-glucosaminyl pseudoaglycone is deacetylated prior to long chain hydrocarbon attachment. Here we present several high-resolution crystal structures of the pseudoaglycone deacetylases from the biosynthetic pathways of teicoplanin and A40926. The cocrystal structure of the teicoplanin pseudoaglycone deacetylase with a fatty acid product provides further insights into the roles of active-site residues, and suggests mechanistic similarities with structurally distinct zinc deacetylases, such as peptidoglycan deacetylase and LpxC. A unique, structurally mobile capping lid, located at the apex of these pseudoaglycone deacetylases, likely serves as a determinant of substrate specificity.

Diversity in the polysaccharide component of lipopolysaccharide (LPS) contributes to the persistence and pathogenesis of Gram-negative bacteria. The Nudix hydrolase GDP-mannose mannosyl hydrolase (Gmm) contributes to this diversity by regulating the concentration of mannose in LPS biosynthetic pathways. Here, we present seven high-resolution crystal structures of Gmm from the enteropathogenic E. coli strain O128: the structure of the apo enzyme, the cocrystal structure of Gmm bound to the product Mg2+-GDP, two cocrystal structures of precatalytic and turnover complexes of Gmm-Ca2+-GDP-alpha-d-mannose, and three cocrystal structures of an inactive mutant (His-124 --> Leu) Gmm bound to substrates GDP-alpha-d-mannose, GDP-alpha-d-glucose, and GDP-beta-l-fucose. These crystal structures help explain the molecular basis for substrate specificity and promiscuity and provide a structural framework for reconciling previously determined kinetic parameters. Unexpectedly, these structures reveal concerted changes in the enzyme structure that result in the formation of a catalytically competent active site only in the presence of the substrate/product. These structural views of the enzyme may provide a rationale for the design of inhibitors that target the biosynthesis of LPS by pathogenic bacteria.