Venus Flytrap Domain Research Articles

Ultrasensitive Bioelectronic Tongue Based on the Venus Flytrap Domain of a Human Sweet Taste Receptor.

Sweet taste is an important factor that regulates calorie intake and contributes to food preferences in humans and animals. Therefore, the evaluation of sweet substances is essential for various fields such as healthcare, food, and pharmaceutical industries. Sweet tastants are detected by sweet taste receptors which are class C G-protein-coupled receptors. T1R2 venus flytrap (VFT) of the sweet taste receptor is known as a primary ligand-binding domain for sweet tastants. In this study, we developed an ultrasensitive artificial sweet taste bioelectronic tongue based on the T1R2 VFT of a human sweet taste receptor. Here, the T1R2 VFT of a human sweet taste receptor was successfully overexpressed in a bacterial expression system. A T1R2 VFT-immobilized carbon nanotube field-effect transistor with floating electrodes was exploited as an artificial sweet taste sensory system. Significantly, our T1R2 VFT-functionalized bioelectronic tongue could be used to detect solutions of sweet tastants down to 0.1 fM and selectively discriminate sweet substances from other taste substances. Furthermore, our device could be used to monitor the response of the T1R2 VFT domain of a sweet taste receptor to sweet substances in real food environments such as apple juice and chamomile herb tea. Moreover, our device was used to evaluate the inhibition and enhancement effects on sweet taste receptors by zinc ions and chamomile tea, respectively. In addition, our device demonstrated long-term storability and reusability. In this respect, our sweet taste bioelectronic tongue could be a promising tool for various basic research and industrial applications.

Sweet taste perception in mice is blunted by PTBP1-regulated skipping of Tas1r2 exon 4.

Taste perception, initiated by activation of taste receptors in taste bud cells, is crucial for regulating nutrient intake. Genetic polymorphisms in taste receptor genes cannot fully explain the wide individual variations of taste sensitivity. Alternative splicing (AS) is a ubiquitous posttranscriptional mode of gene regulation that enriches the functional diversity of proteins. Here, we report the identification of a novel splicing variant of sweet taste receptor gene Tas1r2 (Tas1r2_∆e4) in mouse taste buds and the mechanism by which it diminishes sweet taste responses in vitro and in vivo. Skipping of Tas1r2 exon 4 in Tas1r2_∆e4 led to loss of amino acids in the extracellular Venus flytrap domain, and the truncated isoform reduced the response of sweet taste receptors (STRs) to all sweet compounds tested by generating nonfunctional T1R2/T1R3 STR heterodimers. The splicing factor PTBP1 (polypyrimidine tract-binding protein 1) promoted Tas1r2_∆e4 generation through binding to a polypyrimidine-rich splicing silencer in Tas1r2 exon 4, thus decreasing STR function and sweet taste perception in mice. Taken together, these data reveal the existence of a regulated AS event in Tas1r2 expression and its effect on sweet taste perception, providing a novel mechanism for modulating taste sensitivity at the posttranscriptional level.

Investigating mechanism of sweetness intensity differences through dynamic analysis of sweetener–T1R2–membrane systems

Knowing the mechanism of action of sweet taste receptors is important for the design of new, healthy sweeteners. However, little is known about the structures and recognition mechanisms of these receptors. 28 sweeteners were assessed by molecular docking, and 8 typical sweeteners were chosen to construct sweetener–T1R2–membrane systems to analyze interactions between receptor and sweeteners. Natural sweeteners with low-intensity sweetness, such as fructose and xylitol, were released from the Venus flytrap domain at ∼30 ns, with displacements greater than 50 Å. In contrast, artificial neotame and advantame bound stably to the receptor, within 5 Å. Van der Waals interactions were significant in high-intensity sweetener systems, imparting an energy difference of over 15 kcal/mol between neotame (artificial sweetener) and fructose (natural). These results provide a deeper understanding of the mechanisms of sweetener function and offer a new direction for the design of sweeteners.

Predicted Structure of Fully Activated Tas1R3/1R3' Homodimer Bound to G Protein and Natural Sugars: Structural Insights into G Protein Activation by a Class C Sweet Taste Homodimer with Natural Sugars.

The Tas1R3 G protein-coupled receptor constitutes the main component of sweet taste sensory response in humans via forming a heterodimer with Tas1R2 or a homodimer with Tas1R3. The Tas1R3/1R3' homodimer serves as a low-affinity sweet taste receptor, stimulating gustducin G protein (GGust) signaling in the presence of a high concentration of natural sugars. This provides an additional means to detect the taste of natural sugars, thereby differentiating the flavors between natural sugars and artificial sweeteners. We report here the predicted 3D structure of active state Tas1R3/1R3' homodimer complexed with heterotrimeric GGust and sucrose. We discovered that the GGust makes ionic anchors to intracellular loops 1 and 2 of Tas1R3 while the Gα-α5 helix engages the cytoplasmic region extensively through salt bridge and hydrophobic interactions. We show that in the activation of this complex the Venus flytrap domains of the homodimer undergo a remarkable twist up to ∼100° rotation around the vertical axis to adopt a closed-closed conformation while the intracellular region relaxes to an open-open conformation. We find that binding of sucrose to the homodimer stabilizes a preactivated conformation with a largely open intracellular region that recruits and activates the GGust. Upon activation, the Gα subunit spontaneously opens up the nucleotide-binding site, making nucleotide exchange facile for signaling. This activation of GGust promotes the interdomain twist of the Venus flytrap domains. These structures and transformations could potentially be a basis for the design of new sweeteners with higher activity and less unpleasant flavors.

Human-like performance umami electrochemical biosensor by utilizing co-electrodeposition of ligand binding domain T1R1-VFT and Prussian blue

Over the past decades, due to the desire for artificial umami flavors, apparatuses for detecting the umami taste have constantly been developed. Nevertheless, most information on umami is still acquired through human sensory assessment, which makes it difficult to establish an umami standard or quantify the umami flavor. In this study, the ligand binding domain called venus flytrap (VFT) domain of the umami taste receptor protein T1R1 was used as a recognition element, and an electrochemical biosensor based on a double-signal amplification strategy was constructed using single-walled carbon nanotubes (SWCNTs) and Prussian blue (PB). Moreover, the umami taste of four representative umami substances, inosine-5′-monophosphate (IMP), monosodium L-glutamate (MSG), beefy meaty peptide (BMP), and sodium succinate (WSA), were successfully quantitatively measured using differential pulse voltammetry (DPV) at an electrochemical workstation. Based on an equation (S/N = 3), the low detection limits (LODs) of IMP, MSG, BMP, and WSA were 0.1, 0.1, 0.1, and 0.01 pM, respectively. Meanwhile, a normalized signal intensity of more than 90% was kept for 4 days. The results showed that the biosensor could be used to detect umami substances with high sensitivity and selectivity, and was shown to have human-like performance. To develop the T1R1-VFT biosensor using the above-mentioned method, we utilized the ligand binding domain of the human umami receptor, rather than the entire umami receptor protein, which had a complex structure, having the following advantages: volume reduction, simplicity, and stability. This method has great potential for the detection of umami tastes, instead of using sensory evaluation, and for the development of new artificial flavorings.

Metabotropic glutamate receptors in GtoPdb v.2021.3

Metabotropic glutamate (mGlu) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Metabotropic Glutamate Receptors [347]) are a family of G protein-coupled receptors activated by the neurotransmitter glutamate [138]. The mGlu family is composed of eight members (named mGlu1 to mGlu8) which are divided in three groups based on similarities of agonist pharmacology, primary sequence and G protein coupling to effector: Group-I (mGlu1 and mGlu5), Group-II (mGlu2 and mGlu3) and Group-III (mGlu4, mGlu6, mGlu7 and mGlu8) (see Further reading).Structurally, mGlu are composed of three juxtaposed domains: a core G protein-activating seven-transmembrane domain (TM), common to all GPCRs, is linked via a rigid cysteine-rich domain (CRD) to the Venus Flytrap domain (VFTD), a large bi-lobed extracellular domain where glutamate binds. mGlu form constitutive dimers, cross-linked by a disulfide bridge. The structures of the VFTD of mGlu1, mGlu2, mGlu3, mGlu5 and mGlu7 have been solved [198, 271, 264, 399]. The structure of the 7 transmembrane (TM) domains of both mGlu1 and mGlu5 have been solved, and confirm a general helical organization similar to that of other GPCRs, although the helices appear more compacted [87, 429, 61]. Recent advances in cryo-electron microscopy have provided structures of full-length mGlu receptor dimers [189]. Studies have revealed the possible formation of heterodimers between either group-I receptors, or within and between group-II and -III receptors [88]. First well characterized in transfected cells, co-localization and specific pharmacological properties also suggest the existence of such heterodimers in the brain [266].[436, 143, 279]. Beyond heteromerization with other mGlu receptor subtypes, increasing evidence suggests mGlu receptors form heteromers and larger order complexes with class A GPCRs (reviewed in [138]). The endogenous ligands of mGlu are L-glutamic acid, L-serine-O-phosphate, N-acetylaspartylglutamate (NAAG) and L-cysteine sulphinic acid. Group-I mGlu receptors may be activated by 3,5-DHPG and (S)-3HPG [30] and antagonized by (S)-hexylhomoibotenic acid [232]. Group-II mGlu receptors may be activated by LY389795 [265], LY379268 [265], eglumegad [350, 430], DCG-IV and (2R,3R)-APDC [351], and antagonised by eGlu [168] and LY307452 [421, 103]. Group-III mGlu receptors may be activated by L-AP4 and (R,S)-4-PPG [128]. An example of an antagonist selective for mGlu receptors is LY341495, which blocks mGlu2 and mGlu3 at low nanomolar concentrations, mGlu8 at high nanomolar concentrations, and mGlu4, mGlu5, and mGlu7 in the micromolar range [183]. In addition to orthosteric ligands that directly interact with the glutamate recognition site, allosteric modulators that bind within the TM domain have been described. Negative allosteric modulators are listed separately. The positive allosteric modulators most often act as ‘potentiators’ of an orthosteric agonist response, without significantly activating the receptor in the absence of agonist.

Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.

SummaryMetabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.

In silico identification of novel small molecule umami peptide from ovotransferrin

SummaryUmami ingredients are the important components of seasoning in food and widely used in food production. However, few novel umami peptides were isolated and identified. Here, the purpose of this research is to identify small molecule umami peptides derived from ovotransferrin by molecular docking. Based on the virtual enzymatic hydrolysis and molecular docking analysis, the tripeptide DEK with the lowest CDOCKER‐ENERGY (−99.50 kcal mol−1) was identified, which could occupy the extracellular venus flytrap domain (VFTD) of T1R1/T1R3 (taste receptor type 1, member 1 and 3). Furthermore, the electronic tongue was used to measure the umami taste of DEK. The results showed that the umami value of DEK was 2.31, which was higher than that of monosodium L‐glutamate via electronic tongue analysis. The docking results showed that virtual screening and molecular docking may be a more reliable and rapid method to substitute for classical methods for screening and identification of small molecule umami peptides.

Novel umami peptides from tilapia lower jaw and molecular docking to the taste receptor T1R1/T1R3

This study aimed to isolate and identify peptides with intense umami taste from tilapia lower jaw. The aqueous extract was separated using ultrafiltration and Sephadex G-15 gel filtration chromatography. The peptide fraction with an intense umami taste was selected by sensory evaluation. The five novel peptides with strong umami taste were VADLMR, STELFK, FVGLQER, DALKKK, and VVLNPVARVE. Electronic tongue analysis and sensory evaluation showed that five peptides had obvious umami taste characteristics, and the recognition thresholds of umami peptides were in the range 0.125–0.250 mg/mL. Molecular docking was used to study the interaction of the peptides and umami taste receptor T1R1/T1R3. The five peptides could perfectly be inserted into the binding pocket of the Venus flytrap domain in the T1R3 subunit. Hydrogen bonding and hydrophobic interaction were the important interaction forces. The five peptides may bind with Asp219, Glu217, and Glu148 in T1R1/T1R3 receptor and produce the umami taste.

Probing The Structural Basis of Metabotropic Glutamate Receptor 5 (mGlu 5 ) Activation and Positive Allosteric Modulation

The metabotropic glutamate receptor 5 (mGlu5) is a class C G protein-coupled receptor (GPCR) that has emerged as a promising drug target for multiple central nervous system disorders. Structurally, mGlu5 forms obligate dimers. Each protomer possesses a 7-transmembrane (7TM) domain which is connected through a cysteine-rich domain (CRD) to a large extracellular domain (ECD), encompassing the orthosteric ligand-binding Venus flytrap domain (VFD). Recent spectroscopic studies on mGlu5 signaling demonstrated large-scale conformational changes underlie signal transmission from the ECD to the G protein coupling 7TM domain. Although these studies reported the necessity of extracellular loops (ECL) for activation of mGlu5 by orthosteric agonists, specific residues related to transmission of receptor activation remain unknown. We used a combination of site-directed mutagenesis, Ca2+ mobilization assays and radioligand binding studies to identify residues within the ECLs that aid in the propagation of orthosteric agonist-induced conformational changes in the VFD to the 7TM. Multiple ECL2 residues markedly reduced activation by glutamate but not allosteric agonists. These same mutations had no effect on receptor expression and thus likely contribute to the mGlu­5 activation network. Select mutations that compromised glutamate activation could be rescued with positive allosteric modulators (PAMs). mGlu5 PAMs are promising potential new therapeutic strategies for schizophrenia and cognition, however, structure-activity relationships for mGlu5 PAMs are notoriously difficult to interpret. A deeper understanding of the structural basis of dynamic receptor activation and modulation processes will aid future rational structure-based drug discovery efforts

Taste characteristics and umami mechanism of novel umami peptides and umami-enhancing peptides isolated from the hydrolysates of Sanhuang Chicken

This study identified umami and umami-enhancing peptides, which explain the umami taste of Sanhuang chicken. Ten novel peptides were isolated from chicken hydrolysate by gel filtration chromatography and reverse-phase high-performance liquid chromatography. The amino acid sequence was analyzed by UPLC-Q-Exactive Orbitrap-MS: HGEDKEGE, IPIPATKT, SEASNNK, IGPGLGR, AGDDAPR, FAGDDAPR, ADGLWL, KDGGGGK, PGPAGPAGP, and GFLGPQ. All synthetic peptides were discovered with umami and umami-enhancing effects based on the sensory evaluation and electronic tongue. Of these, HGEDKEGE, and SEASNNK showed a prominent umami taste, and the umami threshold values of the umami peptide were 0.56 and 0.33 mmol/L, respectively. The AGDDAPR and IGPGLGR had better a umami-enhancing effect. The molecular docking results confirmed that all peptides entered the Venus flytrap (VFT) domain of T1R3, new binding sites were obtained, Arg303, Ser123, and His121 might play critical roles in the umami-enhancing effects. Residues of Ser146 and Ser123 were found to play decisive roles in umami taste.

Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca2+ ions and L-tryptophan

The human calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) responsible for maintaining Ca2+ homeostasis in the blood. The general consensus is that extracellular Ca2+ is the principal agonist of CaSR. Aliphatic and aromatic L-amino acids, such as L-Phe and L-Trp, increase the sensitivity of CaSR towards Ca2+ and are considered allosteric activators. Crystal structures of the extracellular domain (ECD) of CaSR dimer have demonstrated Ca2+ and L-Trp binding sites and conformational changes of the ECD upon Ca2+/L-Trp binding. However, it remains to be understood at the structural level how Ca2+/L-Trp binding to the ECD leads to conformational changes in transmembrane domains (TMDs) and consequent CaSR activation. Here, we determined the structures of full-length human CaSR in the inactive state, Ca2+- or L-Trp-bound states, and Ca2+/L-Trp-bound active state using single-particle cryo-electron microscopy. Structural studies demonstrate that L-Trp binding induces the closure of the Venus flytrap (VFT) domain of CaSR, bringing the receptor into an intermediate active state. Ca2+ binding relays the conformational changes from the VFT domains to the TMDs, consequently inducing close contact between the two TMDs of dimeric CaSR, activating the receptor. Importantly, our structural and functional studies reveal that Ca2+ ions and L-Trp activate CaSR cooperatively. Amino acids are not able to activate CaSR alone, but can promote the receptor activation in the presence of Ca2+. Our data provide complementary insights into the activation of class C GPCRs and may aid in the development of novel drugs targeting CaSR.

Les avancées récentes dans le domaine de la biologie structurale des récepteurs couplés aux protéines G de la classe C : Le récepteur métabotropique du glutamate 5

Class C GPCRs, that include metabotropic glutamate receptors (mGlu), taste receptors, GABAB receptor and Calcium-sensing receptor, are unusual in terms of their molecular architecture and allosteric regulation. They all form obligatory dimers, dimerization being fundamental for their function. More specifically, the mGlu are activated by the main excitatory neurotransmitter, L-glutamate. mGlu activation by glutamate binding in the venus flytrap domain (VFT) triggers conformational changes that are transmitted, through the Cystein-Rich Domain (CRD), to the conserved fold of 7 transmembrane helices (7TM), that couples to intracellular G protein. mGlu activity can also be allosterically modulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7TM. Recent progress in cryo-electron microscopy (cryoEM) has allowed unprecedented advances in deciphering the structural and molecular basis of their activation mechanism. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. The diversity of inactive conformations for the class C was unexpected but allows PAM stabilising a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. Here we present and discuss recent structural characterisation of mGlu receptors, highlighting findings that make the class C of GPCR unique. Understanding the structural basis of mGlu dimer signaling represents a landmark achievement and paves the way for structural investigation of GPCR dimer signaling in general. Structural information will open new avenues for structure-based drug design.

Cell Surface Calcium-Sensing Receptor Heterodimers: Mutant Gene Dosage Affects Ca2+ Sensing but Not G Protein Interaction.

ABSTRACTThe calcium‐sensing receptor is a homodimeric class C G protein‐coupled receptor (GPCR) that senses extracellular Ca2+ (Ca2+ o) via a dimeric extracellular Venus flytrap (VFT) unit that activates G protein‐dependent signaling via twin Cysteine‐rich domains linked to transmembrane heptahelical (HH) bundles. It plays a key role in the regulation of human calcium and thus mineral metabolism. However, the nature of interactions between VFT units and HH bundles, and the impacts of heterozygous or homozygous inactivating mutations, which have implications for disorders of calcium metabolism are not yet clearly defined. Herein we generated CaSR‐GABAB1 and CaSR‐GABAB2 chimeras subject to GABAB‐dependent endoplasmic reticulum sorting to traffic mutant heterodimers to the cell surface. Transfected HEK‐293 cells were assessed for Ca2+ o‐stimulated Ca2+ i mobilization using mutations in either the VFT domains and/or HH bundle intraloop‐2 or intraloop‐3. When the same mutation was present in both VFT domains of receptor dimers, analogous to homozygous neonatal severe hyperparathyroidism (NSHPT), receptor function was markedly impaired. Mutant heterodimers containing one wild‐type (WT) and one mutant VFT domain, however, corresponding to heterozygous familial hypocalciuric hypercalcemia type‐1 (FHH‐1), supported maximal signaling with reduced Ca2+ o potency. Thus two WT VFT domains were required for normal Ca2+ o potency and there was a pronounced gene‐dosage effect. In contrast, a single WT HH bundle was insufficient for maximal signaling and there was no functional difference between heterodimers in which the mutation was present in one or both intraloops; ie, no gene‐dosage effect. Finally, we observed that the Ca2+ o‐stimulated CaSR operated exclusively via signaling in‐trans and not via combined in‐trans and in‐cis signaling. We consider how receptor asymmetry may support the underlying mechanisms. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Identification, taste characterization and molecular docking study of novel umami peptides from the Chinese anchovy sauce.

Fish sauce has a subtle flavor with prominent umami and salty taste, and is accompanied by a certain sweetness and bitterness. In order to identify a wider range of umami peptides, Chinese southern and northern anchovy sauce were selected for the study. Seventeen peptides were obtained by separation and purification, and their taste activity was predicted. Through the taste characterization and descriptive analysis, it was found that the synthesized peptides were umami and umami-enhancing peptides. Seventeen umami peptides were simulated and embedded into the umami receptor T1R1/T1R3 by inserting into the Venus flytrap domain (VFTD) of the T1R3 subunit; the interaction forces were mainly hydrogen bonding, electrostatic interaction, van der Waals force and hydrophobic interaction. According to the docking interaction energies, long-chain peptides may be easier to bind to the receptor than short-chain peptides. Asp196, Glu128 and Glu197 were the main binding sites for docking, and could affect umami synergism. For the first time, novel umami peptides in Chinese anchovy sauce have been reported. This study is helpful for discovering umami marine resource peptides, and can provide a basis for further understanding the flavor system of anchovy sauce. © 2020 Society of Chemical Industry.

The GABAB Receptor—Structure, Ligand Binding and Drug Development

The γ-aminobutyric acid (GABA) type B receptor (GABAB-R) belongs to class C of the G-protein coupled receptors (GPCRs). Together with the GABAA receptor, the receptor mediates the neurotransmission of GABA, the main inhibitory neurotransmitter in the central nervous system (CNS). In recent decades, the receptor has been extensively studied with the intention being to understand pathophysiological roles, structural mechanisms and develop drugs. The dysfunction of the receptor is linked to a broad variety of disorders, including anxiety, depression, alcohol addiction, memory and cancer. Despite extensive efforts, few compounds are known to target the receptor, and only the agonist baclofen is approved for clinical use. The receptor is a mandatory heterodimer of the GABAB1 and GABAB2 subunits, and each subunit is composed of an extracellular Venus Flytrap domain (VFT) and a transmembrane domain of seven α-helices (7TM domain). In this review, we briefly present the existing knowledge about the receptor structure, activation and compounds targeting the receptor, emphasizing the role of the receptor in previous and future drug design and discovery efforts.

Structural basis of the activation of a metabotropic GABA receptor.

Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and are implicated in various neuropsychological conditions, ranging from addiction to psychosis1. GABAB belongs to G protein-coupled receptor class C, and its functional entity consists of an obligate heterodimer composed of GB1 and GB22. Each subunit possesses an extracellular Venus flytrap domain, connected to a canonical seven-transmembrane domain. Here, we present four cryo-EM structures of the human full-length GB1-GB2 heterodimer in its inactive apo, two intermediate agonist-bound, and active agonist/positive allosteric modulator bound forms. The structures reveal startling differences, shedding light onto the complex motions underlying the unique activation mechanism of GABAB. Our results show that agonist binding in the GB1 Venus flytrap domain triggers a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism, potentiated by a positive allosteric modulator binding at the dimerization interface, to initiate downstream signaling.

Cryo-EM structures of inactive and active GABAB receptor

Metabotropic GABAB G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of full-length human heterodimeric GABAB receptor in the antagonist-bound inactive state and in the active state complexed with an agonist and a positive allosteric modulator in the presence of Gi1 protein at a resolution range of 2.8-3.0 Å. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between the two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistic shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of Gi1. We also observed that the positive allosteric modulator anchors at the dimeric interface of TM domains. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting the dimeric interface of GABAB receptor.

Exploring Conformational Dynamics of the Extracellular Venus flytrap Domain of the GABAB Receptor: A Path-Metadynamics Study.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS). Dysfunctional GABAergic neurotransmission is associated with numerous neurological and neuropsychiatric disorders. The GABAB receptor (GABAB-R) is a heterodimeric class C G protein-coupled receptor (GPCR) comprised of GABAB1a/b and GABAB2 subunits. The orthosteric binding site for GABA is located in the extracellular Venus flytrap (VFT) domain of the GABAB1a/b. Knowledge about molecular mechanisms and druggable receptor conformations associated with activation is highly important to understand the receptor function and for rational drug design. Currently, the conformational changes of the receptor upon activation are not well described. On the basis of other class C members, the VFT is proposed to fluctuate between an open/inactive and closed/active state and one of these conformations is stabilized upon ligand binding. In the present study, we investigated the dynamics of the GABAB1b-R VFT in the apo form by combining unbiased molecular dynamics with path-metadynamics. Our simulations confirmed the open/inactive and closed/active state as the main conformations adopted by the receptor. Sizeable energy barriers were found between stable minima, suggesting a relatively slow interconversion. Previously undisclosed metastable states were also identified, which might hold potential for future drug discovery efforts.

Structure-based screening for discovery of sweet compounds

Sweet taste is a cue for calorie-rich food and is innately attractive to animals, including humans. In the context of modern diets, attraction to sweetness presents a significant challenge to human health. Most known sugars and sweeteners bind to the Venus Fly Trap domain of T1R2 subunit of the sweet taste heterodimer. Because the sweet taste receptor structure has not been experimentally solved yet, a possible approach to finding sweet molecules is virtual screening using compatibility of candidate molecules to homology models of sugar-binding site. Here, the constructed structural models, docking and scoring schemes were validated by their ability to rank known sweet-tasting compounds higher than properties-matched random molecules. The best performing models were next used in virtual screening, retrieving recently patented sweeteners and providing novel predictions.