scholarly journals Different conformational responses of the β2-adrenergic receptor-Gs complex upon binding of the partial agonist salbutamol or the full agonist isoprenaline

2020 ◽  
Author(s):  
Fan Yang ◽  
Shenglong Ling ◽  
Yingxin Zhou ◽  
Yanan Zhang ◽  
Pei Lv ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hydrophobic Interactions ◽  
Partial Agonist ◽  
Binding Pocket ◽  
Structural Basis ◽  
Spectroscopic Techniques ◽  
Toggle Switch ◽  
Intracellular Loop ◽  
Full Agonist ◽  
Β2 Adrenergic Receptor

Abstract GPCRs are responsible for most cytoplasmic signaling in response to extracellular ligands with different efficacy profiles. Various spectroscopic techniques have identified that agonists exhibiting varying efficacies can selectively stabilize a specific conformation of the receptor. However, the structural basis for activation of the GPCR-G protein complex by ligands with different efficacies is incompletely understood. To better understand the structural basis underlying the mechanisms by which ligands with varying efficacies differentially regulate the conformations of receptors and G proteins, we determined the structures of β2AR-Gαs$\beta $γ bound with partial agonist salbutamol or bound with full agonist isoprenaline using single-particle cryo-electron microscopy at resolutions of 3.26 Å and 3.80 Å, respectively. Structural comparisons between the β2AR-Gs-salbutamol and β2AR-Gs-isoprenaline complexes demonstrated that the decreased binding affinity and efficacy of salbutamol compared with those of isoprenaline might be attributed to the weakened hydrogen bonding interactions, attenuated hydrophobic interactions in the orthosteric binding pocket and different conformational changes in the rotamer toggle switch in TM6. Moreover, the observed stronger interactions between the intracellular loop 2 or 3 (ICL2 or ICL3) of β2AR and Gαs with the binding of salbutamol versus isoprenaline might decrease phosphorylation in the salbutamol-activated β2AR-Gs complex. From the observed structural differences between these complexes of β2AR, a mechanism of β2AR activation by partial and full agonists is proposed to shed structural insights for β2AR desensitization.

scholarly journals Structural basis of rifampin inactivation by rifampin phosphotransferase

2016 ◽  
Vol 113 (14) ◽  
pp. 3803-3808 ◽  
Author(s):  
Xiaofeng Qi ◽  
Wei Lin ◽  
Miaolian Ma ◽  
Chengyuan Wang ◽  
Yang He ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Conformational Changes ◽  
Bacterial Infections ◽  
Binding Pocket ◽  
Resistance Mechanisms ◽  
Binding Domain ◽  
Structural Basis ◽  
Atp Binding ◽  
Toggle Switch ◽  
The Cost

Rifampin (RIF) is a first-line drug used for the treatment of tuberculosis and other bacterial infections. Various RIF resistance mechanisms have been reported, and recently an RIF-inactivation enzyme, RIF phosphotransferase (RPH), was reported to phosphorylate RIF at its C21 hydroxyl at the cost of ATP. However, the underlying molecular mechanism remained unknown. Here, we solve the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations. LmRPH comprises three domains: an ATP-binding domain (AD), an RIF-binding domain (RD), and a catalytic His-containing domain (HD). Structural analyses reveal that the C-terminal HD can swing between the AD and RD, like a toggle switch, to transfer phosphate. In addition to its catalytic role, the HD can bind to the AD and induce conformational changes that stabilize ATP binding, and the binding of the HD to the RD is required for the formation of the RIF-binding pocket. A line of hydrophobic residues forms the RIF-binding pocket and interacts with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moieties of RIF. The R group of RIF points toward the outside of the pocket, explaining the low substrate selectivity of RPH. Four residues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and Gln337, were found to play essential roles in the phosphorylation of RIF; among these the His825 residue may function as the phosphate acceptor and donor. Our study reveals the molecular mechanism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins.

scholarly journals Structural basis for subtype-specific inhibition of the P2X7 receptor

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Akira Karasawa ◽  
Toshimitsu Kawate
Keyword(s):  
P2x7 Receptor ◽  
Hydrophobic Interactions ◽  
Binding Pocket ◽  
Drug Binding ◽  
Structural Basis ◽  
Atp Binding ◽  
Allosteric Site ◽  
Channel Opening ◽  
A Site ◽  
Novel Drug

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

Structural basis of the transactivation deficiency of the human PPARγ F360L mutant associated with familial partial lipodystrophy

2014 ◽  
Vol 70 (7) ◽  
pp. 1965-1976 ◽  
Author(s):  
Clorinda Lori ◽  
Alessandra Pasquo ◽  
Roberta Montanari ◽  
Davide Capelli ◽  
Valerio Consalvi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Partial Agonist ◽  
Salt Bridge ◽  
Van Der Waals Interactions ◽  
Structural Basis ◽  
Wild Type ◽  
Partial Lipodystrophy ◽  
X Ray ◽  
Familial Partial Lipodystrophy

The peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate glucose and lipid metabolism. The role of PPARs in several chronic diseases such as type 2 diabetes, obesity and atherosclerosis is well known and, for this reason, they are the targets of antidiabetic and hypolipidaemic drugs. In the last decade, some rare mutations in human PPARγ that might be associated with partial lipodystrophy, dyslipidaemia, insulin resistance and colon cancer have emerged. In particular, the F360L mutant of PPARγ (PPARγ2 residue 388), which is associated with familial partial lipodystrophy, significantly decreases basal transcriptional activity and impairs stimulation by synthetic ligands. To date, the structural reason for this defective behaviour is unclear. Therefore, the crystal structure of PPARγ F360L together with the partial agonist LT175 has been solved and the mutant has been characterized by circular-dichroism spectroscopy (CD) in order to compare its thermal stability with that of the wild-type receptor. The X-ray analysis showed that the mutation induces dramatic conformational changes in the C-terminal part of the receptor ligand-binding domain (LBD) owing to the loss of van der Waals interactions made by the Phe360 residue in the wild type and an important salt bridge made by Arg357, with consequent rearrangement of loop 11/12 and the activation function helix 12 (H12). The increased mobility of H12 makes the binding of co-activators in the hydrophobic cleft less efficient, thereby markedly lowering the transactivation activity. The spectroscopic analysis in solution and molecular-dynamics (MD) simulations provided results which were in agreement and consistent with the mutant conformational changes observed by X-ray analysis. Moreover, to evaluate the importance of the salt bridge made by Arg357, the crystal structure of the PPARγ R357A mutant in complex with the agonist rosiglitazone has been solved.

scholarly journals Structural Basis of Vitamin K Antagonism

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 482-482
Author(s):  
Weikai Li ◽  
Shixuan Liu ◽  
Shuang Li
Keyword(s):  
Vitamin K ◽  
Conformational Changes ◽  
Conflicts Of Interest ◽  
Bone Mineralization ◽  
Vitamin K Antagonists ◽  
Membrane Surface ◽  
Warfarin Dose ◽  
Binding Pocket ◽  
Structural Basis ◽  
Vitamin K Cycle

The vitamin K cycle supports blood coagulation, bone mineralization, and vascular calcium homeostasis. A key enzyme in this cycle, vitamin K epoxide reductase (VKOR), is the target of vitamin K antagonists (VKAs). Despite their extensive clinical use, the dose of VKAs (e.g., warfarin) is hard to regulate and overdose can lead to fatal bleeding. Improving the dose regulation requires understanding how VKAs inhibit VKOR, which is a membrane-embedded enzyme difficult to characterize with structural and biochemical studies. Here we achieve a long-standing goal of obtaining crystal structures of human VKOR with warfarin, which represents coumarin-based VKAs; with phenindione, which represents indandione-based VKAs; with superwarfarins, the most commonly used rodenticides; and with vitamin K epoxide in a reaction intermediate state. We have also solved structures of a VKOR-like homolog with warfarin, with vitamin K substrates, and without ligand. These structures show that human VKOR adopts an overall fold with four transmembrane helices (TM) and a large ER-luminal region. VKAs are bound at the active site of HsVKOR, which is formed by the surrounding four-TM bundle and a cap domain on top. The cap domain is stabilized by a linked anchor domain that interacts with the membrane surface. VKOR binds specifically to VKAs through hydrogen bonding to their diketone groups. Mutating VKOR residues recognizing the diketones render strong warfarin resistance. Except the hydrogen bonds, the binding pocket is largely hydrophobic. This pocket is incompatible with warfarin metabolite, explaining the inactivation of warfarin through CYP2C9 metabolism; CYP2C9 and VKOR genotypes can explain 30-50% of the patient variability in warfarin dose. In addition, the high potency of superwarfarins is due to the interaction of their side group with a tunnel where the isoprenyl chain of vitamin K is bound. For VKOR catalysis, the same residues affording the VKA-binding specificity also facilitate substrate reduction Initiation of the catalysis requires a reactive cysteine to form a substrate adduct. Interactions from this stably bound adduct induces a closed conformation, thereby triggering electron transfer to reduce the substrate. Importantly, the open to closed conformational change during catalysis similar to that induced by the binding of VKAs. Taken together, VKAs achieve inhibition through mimicking key interactions and conformational changes required for VKOR catalytic cycle. Understanding of these mechanisms will enable improved strategy to regulate warfarin dose and have a broad impact on thromboembolic diseases and bone disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rhodopsin: Structural Basis of Molecular Physiology

2001 ◽  
Vol 81 (4) ◽  
pp. 1659-1688 ◽  
Author(s):  
Santosh T. Menon ◽  
May Han ◽  
Thomas P. Sakmar
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Critical Evaluation ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Structural Basis ◽  
Molecular Physiology ◽  
Movement Model ◽  
Ligand Binding Pocket ◽  
Cytoplasmic Surface

The crystal structure of rod cell visual pigment rhodopsin was recently solved at 2.8-Å resolution. A critical evaluation of a decade of structure-function studies is now possible. It is also possible to begin to explain the structural basis for several unique physiological properties of the vertebrate visual system, including extremely low dark noise levels as well as high gain and color detection. The ligand-binding pocket of rhodopsin is remarkably compact, and several apparent chromophore-protein interactions were not predicted from extensive mutagenesis or spectroscopic studies. The transmembrane helices are interrupted or kinked at multiple sites. An extensive network of interhelical interactions stabilizes the ground state of the receptor. The helix movement model of receptor activation, which might apply to all G protein-coupled receptors (GPCRs) of the rhodopsin family, is supported by several structural elements that suggest how light-induced conformational changes in the ligand-binding pocket are transmitted to the cytoplasmic surface. The cytoplasmic domain of the receptor is remarkable for a carboxy-terminal helical domain extending from the seventh transmembrane segment parallel to the bilayer surface. Thus the cytoplasmic surface appears to be approximately the right size to bind to the transducin heterotrimer in a one-to-one complex. Future high-resolution structural studies of rhodopsin and other GPCRs will form a basis to elucidate the detailed molecular mechanism of GPCR-mediated signal transduction.

scholarly journals Mechanism of gating and partial agonist action in the glycine receptor

2019 ◽  
Author(s):  
Jie Yu ◽  
Hongtao Zhu ◽  
Remigijus Lape ◽  
Timo Greiner ◽  
Rezvan Shahoei ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Open Channel ◽  
Partial Agonist ◽  
Glycine Receptor ◽  
Md Simulations ◽  
Binding Pocket ◽  
Closed Channel ◽  
Agonist Action ◽  
Receptor Family

SummaryThe glycine receptor is a pentameric, neurotransmitter-activated ion channel that transitions between closed/resting, open and desensitized states. Glycine, a full agonist, produces an open channel probability (Po) of ∼1.0 while partial agonists, such as taurine and γ-amino butyric acid (GABA) yield submaximal Po values. Despite extensive studies of pentameric Cys-loop receptors, there is little knowledge of the molecular mechanisms underpinning partial agonist action and how the receptor transitions from the closed to open and to desensitized conformations. Here we use electrophysiology and molecular dynamics (MD) simulations, together with a large ensemble of single particle cryo-EM reconstructions, to show how agonists populate agonist-bound yet closed channel states, thus explaining their lesser efficacy, yet also populate agonist-bound open and desensitized states. Measurements within the neurotransmitter binding pocket, as a function of bound agonist, provide a metric to correlate the extent of agonist-induced conformational changes to open channel probability across the Cys-loop receptor family.

Langevin Dynamics of Hinge-Motion in Proteins

2000 ◽  
Author(s):  
Gang Bao ◽  
Shannon Stott
Keyword(s):  
Blood Vessel ◽  
Conformational Changes ◽  
Hydrophobic Interactions ◽  
Binding Pocket ◽  
Biochemical Responses ◽  
3D Geometry ◽  
Electrostatic And Hydrophobic Interactions ◽  
Selectin Ligand ◽  
Stressful Environments ◽  
Ligand Bond

Abstract Many proteins function in mechanically stressful environments. For example, shear flow in blood vessel may exert a force as high as hundreds of piconewtons on an individual selectin-ligand bond. With such a mechanical force, the receptor (selectin) may deform, thereby altering the conformational match between the receptor and the ligand. Although van der Waals forces, electrostatic and hydrophobic interactions are all important, it is often the 3D geometry local to the binding pocket that dictates the characteristics of the bond between the receptor and the ligand. Good conformational matches lead to strong and long-lasting bonds, whereas poor conformational matches do the converse. it is conceivable that conformational changes in proteins in response to various forces serve as a mechanism for transducing mechanical signals into biochemical responses.

scholarly journals Comparison of Taurine- and Glycine-induced Conformational Changes in the M2-M3 Domain of the Glycine Receptor

2004 ◽  
Vol 279 (19) ◽  
pp. 19559-19565 ◽  
Author(s):  
Nian-Lin R. Han ◽  
John D. Clements ◽  
Joseph W. Lynch
Keyword(s):  
Conformational Changes ◽  
Reaction Rate ◽  
Partial Agonist ◽  
Glycine Receptor ◽  
Reaction Rates ◽  
Structural Basis ◽  
Surface Accessibility ◽  
Partial Agonists ◽  
Substituted Cysteine Accessibility ◽  
Receptor Family

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg271to Lys276in the M2-M3 domain of the homomeric α1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.

scholarly journals β-arrestin–biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction

2016 ◽  
Vol 113 (28) ◽  
pp. E4107-E4116 ◽  
Author(s):  
Richard Carr ◽  
Justin Schilling ◽  
Jianliang Song ◽  
Rhonda L. Carter ◽  
Yang Du ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Similar Efficacy ◽  
Intracellular Loop ◽  
Inotropic Effects ◽  
Relative Contribution ◽  
Biased Signaling ◽  
Β2 Adrenergic Receptor ◽  
Β Blocker ◽  
Β Blockers ◽  
Biased Agonist

β-adrenergic receptors (βARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent β1AR and Gi-dependent β2AR pathways leads to enhanced cardiomyocyte death, reduced β1AR expression, and decreased inotropic reserve. β-blockers act to block excessive catecholamine stimulation of βARs to decrease cellular apoptotic signaling and normalize β1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein–dependent signaling, β-arrestin–dependent signaling promotes cardiomyocyte survival. Given that β2AR expression is unaltered in CHF, a β-arrestin–biased agonist that operates through the β2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective β-blocker, has been classified as a β-arrestin–biased agonist that can inhibit basal signaling from βARs and also stimulate cell survival signaling pathways. To understand the relative contribution of β-arrestin bias to the efficacy of select β-blockers, a specific β-arrestin–biased pepducin for the β2AR, intracellular loop (ICL)1–9, was used to decouple β-arrestin–biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1–9 was able to promote β2AR phosphorylation, β-arrestin recruitment, β2AR internalization, and β-arrestin–biased signaling. Interestingly, ICL1–9 was also able to induce β2AR- and β-arrestin–dependent and Ca2+-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1–9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the β2AR and serves as a model for the next generation of cardiovascular drug development.

scholarly journals Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites

2007 ◽  
Vol 402 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Brian J. Holleran ◽  
Marie-Eve Beaulieu ◽  
Christophe D. Proulx ◽  
Pierre Lavigne ◽  
Emanuel Escher ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Partial Agonist ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Urotensin Ii ◽  
Agonist Binding ◽  
Partial Agonists

The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3–17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.

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