Fully activated structure of the sterol-bound Smoothened GPCR-Gi protein complex.

Smoothened (SMO) is an oncoprotein and signal transducer in the Hedgehog signaling pathway that regulates cellular differentiation and embryogenesis. As a member of the Frizzled (Class F) family of G protein-coupled receptors (GPCRs), SMO biochemically and functionally interacts with Gi family proteins. However, key molecular features of fully activated, G protein-coupled SMO remain elusive. We present the atomistic structure of activated human SMO complexed with the heterotrimeric Gi protein and two sterol ligands, equilibrated at 310 K in a full lipid bilayer at physiological salt concentration and pH. In contrast to previous experimental structures, our equilibrated SMO complex exhibits complete breaking of the pi-cation interaction between R4516.32 and W5357.55, a hallmark of Class F receptor activation. The Gi protein couples to SMO at seven strong anchor points similar to those in Class A GPCRs: intracellular loop 1, intracellular loop 2, transmembrane helix 6, and helix 8. On the path to full activation, we find that the extracellular cysteine-rich domain (CRD) undergoes a dramatic tilt, following a trajectory suggested by positions of the CRD in active and inactive experimental SMO structures. Strikingly, a sterol ligand bound to a shallow transmembrane domain (TMD) site in the initial structure migrates to a deep TMD pocket found exclusively in activator-bound SMO complexes. Thus, our results indicate that SMO interacts with Gi prior to full activation to break the molecular lock, form anchors with Gi subunits, tilt the CRD, and facilitate migration of a sterol ligand in the TMD to an activated position.

The mechanism for ligand activation of the GPCR-G protein complex.

G protein­coupled receptors (GPCRs) activate cellular responses ranging from odorants to neurotransmitters. Binding an agonist leads to activation of a heterotrimeric G protein (GP) that stimulates external signaling. Unfortunately, the mechanism remains unknown. We show for 15 class A GPCRs, including opioids, adrenergics, adenosines, chemokines, muscarinics, cannabinoids, serotonins, and dopamines, that interaction of an inactive GP, including Gs, Gi, Go, G11, and Gq, to the inactive GPCR, containing the intracellular ionic lock between transmembrane (TM) helices 3 and 6, evolves exothermically to form a precoupled GPCR-GP complex with an opened TM3-TM6 and the GP-α5 helix partially inserted into the GPCR but not activated. We show that binding of agonist to this precoupled GPCR-GP complex causes the Gα protein to open into its active form, with the guanosine diphosphate exposed for signaling. This GP-first paradigm provides a strategy for developing selective agonists for GPCRs since it is the pharmacophore for the precoupled GPCR-GP complex that should be used to design drugs.

Association between exercise habits and incident type 2 diabetes mellitus in patients with thyroid cancer: nationwide population-based study.

BACKGROUND: We investigated the association between exercise habits before or after thyroidectomy and incident type 2 diabetes mellitus (T2DM) in patients with thyroid cancer. METHODS: An observational cohort study of 69,526 thyroid cancer patients who underwent thyroidectomy for the treatment of thyroid cancer between 2010 and 2016 was performed using the Korean National Health Information Database. Regular exercise was defined as mid-term or vigorous exercise at least 1 day in a week based on a self-reported questionnaire. Patients were divided into four groups according to exercise habits before and after thyroidectomy: persistent non-exercisers, new exercisers, exercise dropouts, and exercise maintainers. RESULTS: During a median follow-up of 4.5 years, 2,720 (3.91%) patients developed T2DM. The incidence of T2DM per 1,000 person years was lower in patients who performed regular exercise before or after thyroidectomy than in persistent non-exercisers (10.77 in persistent non-exerciser group, 8.28 in new exerciser group, 8.59 in exercise dropout group, and 7.61 in exercise maintainer group). Compared with the persistent non-exerciser group, the new exerciser group (hazard ratio [HR] 0.87, 95% confidence interval [CI] 0.78-0.97), the exercise dropout group (HR 0.81, 95% CI 0.72-0.91), and the exercise maintainer group (HR 0.84, 95% CI 0.76-0.93) had lower risks of incident T2DM. Exercising < 1,500 MET-minutes/week in the exercise maintainer group was associated with a lower risk of incident T2DM compared with persistent non-exercisers (< 500: HR 0.80, 95% CI 0.67-0.96, P = 0.002; 500 to < 1,000: HR 0.81, 95% CI 0.71-0.93, P < 0.001; 1,000 to < 1,500: HR 0.81, 95% CI 0.69-0.94, P < 0.001). CONCLUSIONS: Regular exercise before or after thyroidectomy was associated with a lower risk of incident T2DM in patients with thyroid cancer.

An innovative microcoring technology: A novel approach to acne scar treatment.

BACKGROUND: Acne scars present a complex challenge in dermatology and cosmetics, despite advancements in technological interventions such as fractional lasers, microneedling, and surgical procedures. Effective treatment remains elusive for many individuals. OBJECTIVE: This study aims to evaluate the efficacy of rotational fractional resection using 1 mm diameter rotating scalpels as a primary treatment for icepick and boxcar scars on the cheeks and glabella region. METHODS: Three patients with acne scars underwent a single treatment session of rotational fractional resection. Evaluation occurred at the 2-month post-treatment mark to assess improvements in scar appearance and potential skin-related side effects. RESULTS: Following the treatment, significant improvements were observed in the targeted acne scars. Notable enhancements were noted without major skin-related adverse effects, except for minor suture marks. CONCLUSION: The outcomes of this study underscore the potential of rotational fractional resection as an innovative and effective approach in treating acne scars. This single-session cosmetic procedure shows promise in yielding lasting and quantifiable results, offering a hopeful solution for individuals seeking comprehensive acne scar treatment.

Effectiveness of a media literacy-based smoking prevention program in female adolescents.

OBJECTIVES: To examine the effectiveness of a media literacy-based smoking prevention program based on Ajzen's theory of planned behavior in female adolescents. METHODS: This quasi-experimental study was conducted with female high school students aged 16-17 years in Seoul, Republic of Korea. The program provided eight sessions over 4 weeks. Quantitative data were collected before and after online surveys in an intervention (n = 21) and control (n = 21) groups, and analyzed using mixed analysis of variance. Qualitative data on participation experiences was collected by requesting the participants to answer open-ended questions once a week during the intervention and performing co-occurrence analysis of specific terms in the responses was conducted through text mining. RESULTS: Although the program decreased smoking intention and increased smoking media literacy in the intervention group, there were no significant differences between the groups. Qualitative results obtained from the intervention group showed cognitive and behavioral changes in the perception of the harmfulness of e-cigarettes in the media and the expression of a willingness to overcome the temptation to smoke. CONCLUSIONS: Our findings show that the enhancement of smoking media literacy, specifically by correcting misconceptions regarding e-cigarettes promoted by the new media, contributes smoking prevention in female adolescents. It supports calls for an expanded role of public health professionals in health education at the school level.

Structure and Molecular Mechanism of Signaling for the Glucagon-like Peptide-1 Receptor Bound to Gs Protein and Exendin-P5 Biased Agonist.

The glucagon-like peptide-1 receptor (GLP-1R) is a key regulator of blood glucose and a prime target for the treatment of type II diabetes and obesity with multiple public drugs. Here we present a comprehensive computational analysis of the interactions of the activated GLP-1R-Gs signaling complex with a G protein biased agonist, Exendin P5 (ExP5), which possesses a unique N-terminal sequence responsible for the signal bias. Using a refined all-atom model of the ExP5-GLP-1R-Gs complex in molecular dynamics (MD) simulations, we propose a novel mechanism of conformation transduction in which the unique interaction network of ExP5 N-terminus propagates the binding signal across an array of conserved residues at the transmembrane domain to enhance Gs protein coupling at the cytoplasmic end of the receptor. Our simulations reveal previously unobserved interactions important for activation by ExP5 toward GDP-GTP signaling, providing new insights into the mechanism of class B G protein-coupled receptor (GPCR) signaling. These findings offer a framework for the structure-based design of more effective therapeutics.

Multicenter performance evaluation of the Alinity m CMV assay for quantifying cytomegalovirus DNA in plasma samples.

Monitoring of cytomegalovirus (CMV) viral load is critical for informing treatment decisions in order to prevent the severe health consequences of CMV infection or reactivation of latent CMV in immunocompromised individuals. This first field evaluation examined the analytical and clinical performance of the Alinity m CMV assay. Analytical performance was assessed with a commercially available six-member panel, while the clinical performance evaluation compared the Alinity m CMV assay to the RealTime CMV assay and a laboratory-developed test (LDT) as the test of record at three large hospital-based clinical laboratories. Precision of the Alinity m CMV assay was demonstrated with total standard deviation (SD) between 0.08 and 0.28 Log IU/mL. A total of 457 plasma specimens were tested on the Alinity m CMV assay and compared to the test of record at each site (n = 304 with RealTime CMV and n = 153 with LDT CMV). The Alinity m CMV assay had excellent correlation (correlation coefficient r ≥0.942) in comparison to the RealTime CMV or LDT CMV assays. The mean observed bias ranged from -0.03 to 0.34 Log IU/mL. Median onboard turnaround time of Alinity m CMV was less than 3 h. When the CMV assay is run on the Alinity m system, it has the capacity to shorten time to result and, therefore, to therapy.

The atomistic level structure for the activated human κ-opioid receptor bound to the full Gi protein and the MP1104 agonist.

The kappa opioid receptor (κOR) is an important target for pain therapeutics to reduce depression and other harmful side effects of existing medications. The analgesic activity is mediated by κOR signaling through the adenylyl cyclase-inhibitory family of Gi protein. Here, we report the three-dimensional (3D) structure for the active state of human κOR complexed with both heterotrimeric Gi protein and MP1104 agonist. This structure resulted from long molecular dynamics (MD) and metadynamics (metaMD) simulations starting from the 3.1-Å X-ray structure of κOR-MP1104 after replacing the nanobody with the activated Gi protein and from the 3.5-Å cryo-EM structure of µOR-Gi complex after replacing the 168 missing residues. Using MD and metaMD we discovered interactions to the Gi protein with strong anchors to two intracellular loops and transmembrane helix 6 of the κOR. These anchors strengthen the binding, contributing to a contraction in the binding pocket but an expansion in the cytoplasmic region of κOR to accommodate G protein. These remarkable changes in κOR structure reveal that the anchors are essential for activation.

Mechanism of ß-arrestin recruitment by the µ-opioid G protein-coupled receptor.

Agonists to the µ-opioid G protein-coupled receptor (µOR) can alleviate pain through activation of G protein signaling, but they can also induce ß-arrestin activation, leading to such side effects as respiratory depression. Biased ligands to µOR that induce G protein signaling without inducing ß-arrestin signaling can alleviate pain while reducing side effects. However, the mechanism for stimulating ß-arrestin signaling is not known, making it difficult to design optimum biased ligands. We use extensive molecular dynamics simulations to determine three-dimensional (3D) structures of activated ß-arrestin2 stabilized by phosphorylated µOR bound to the morphine and D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) nonbiased agonists and to the TRV130 biased agonist. For nonbiased agonists, we find that the ß-arrestin2 couples to the phosphorylated µOR by forming strong polar interactions with intracellular loop 2 (ICL2) and either the ICL3 or cytoplasmic region of transmembrane (TM6). Strikingly, Gi protein makes identical strong bonds with these same ICLs. Thus, the Gi protein and ß-arrestin2 compete for the same binding site even though their recruitment leads to much different outcomes. On the other hand, we find that TRV130 has a greater tendency to bind the extracellular portion of TM2 and TM3, which repositions TM6 in the cytoplasmic region of µOR, hindering ß-arrestin2 from making polar anchors to the ICL3 or to the cytosolic end of TM6. This dramatically reduces the affinity between µOR and ß-arrestin2.

Immunogenicity of SARS-CoV-2 Vaccine in Kidney Transplant Recipients: A Cross-Sectional Study in Korea.

Eighty-five Korean kidney transplant recipients who received three doses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine were tested with anti-receptor binding domain (RBD) antibody and neutralizing antibody. High anti-RBD antibody (≥ 100 U/mL) and neutralizing antibody responses (≥ 30%) were detected in 51/85 (60.0%) patients. When we divided the patients with the time from transplantation to vaccination (< 1, 1-2.4, 2.5-4.9, and ≥ 5-year), anti-RBD antibody titers were 3.2 U/mL, 27.8 U/mL, 370.2 U/mL, and 5,094.2 U/mL (P < 0.001) and anti-neutralizing antibody levels were 2.2%, 11.6%, 45.6%, and 93.0% (P < 0.001), respectively. Multivariate analysis revealed increased antibody responses when the time from transplantation to vaccination was five years or longer (odds ratio, 12.0; confidence interval, 2.7-52.8). Korean kidney transplant recipients had suboptimal antibody responses after the third dose of SARS-CoV-2 vaccine. A shorter time from transplantation to vaccination was a risk factor for a low antibody response.

Comparison between polynomial regression and weighted least squares regression analysis for verification of analytical measurement range.

OBJECTIVES: Recently, the linearity evaluation protocol by the Clinical & Laboratory Standards Institute (CLSI) has been revised from EP6-A to EP6-ED2, with the statistical method of interpreting linearity evaluation data being changed from polynomial regression to weighted least squares linear regression (WLS). We analyzed and compared the analytical measurement range (AMR) verification results according to the present and prior linearity evaluation guidelines. METHODS: The verification of AMR of clinical chemistry tests was performed using five samples with two replicates in three different laboratories. After analyzing the same evaluation data in each laboratory by the polynomial regression analysis and WLS methods, results were compared to determine whether linearity was verified across the five sample concentrations. In addition, whether the 90% confidence interval of deviation from linearity by WLS was included in the allowable deviation from linearity (ADL) was compared. RESULTS: A linearity of 42.3-56.8% of the chemistry items was verified by polynomial regression analysis in three laboratories. For analysis of the same data by WLS, a linearity of 63.5-78.3% of the test items was verified where the deviation from linearity of all five samples was within the ADL criteria, and the cases where the 90% confidence interval of all deviation from linearity overlapped the ADL was 78.8-91.3%. CONCLUSIONS: Interpreting AMR verification data by the WLS method according to the newly revised CLSI document EP6-ED2 could reduce laboratory workload, enabling efficient laboratory practice.

Evaluation of Serum Beta-2-Microglobulin Assay on Beckman Coulter Chemistry Analyzer.

BACKGROUND: Beta-2 microglobulin (ß2M) has various clinical usages and is an important prognostic marker for multiple myeloma. Although there are concerns of harmonization between assays, performance evaluation and method comparison reports are rare. Here, we evaluated the performance of Beckman-Coulter ß2M assay. METHODS: The precision and linearity of the Beckman Coulter ß2M assay were evaluated. Beckman-Coulter results were compared to DiaSorin Liaison results. The manufacturer provided reference interval was verified. RESULTS: Within-laboratory imprecision percentage coefficient of variation was 1.73% and 2.19% for low- and high-level control material, respectively. The linearity was confirmed over a range of 0.08 to 15.60 mg/L. Passing-Bablok regression analysis showed that y (Beckman-Coulter) = 0.978 x (Liaison) + 0.079 (r = 0.996) with a mean difference of 1.0%. All healthy subjects were within the range of manufacturer provided reference interval. CONCLUSIONS: The analytical performance of the Beckman-Coulter ß2M assay was clinically acceptable.

Performance of the New RAPIDPoint 500e Blood Gas Analyzer and its Comparison with Central Laboratory Biochemistry Analyzer.

BACKGROUND: Blood gas analyzers are commonly used in point of care settings in hospitals to assess and monitor critically ill patients. The blood gas analyzer, RAPIDPoint 500e (Siemens Healthcare, UK), has been newly introduced. Here, we evaluated the performance of RAPIDPoint 500e for the determination of pH, partial carbon dioxide pressure, partial oxygen pressure, sodium (Na+), potassium (K+), chloride (Cl-), ionized calcium, glucose, and lactate. METHODS: Repeatability, within-laboratory imprecision, and linearity were assessed according to CLSI guidelines. RAPIDPoint 500e results were compared to its previous version RAPIDPoint 500. For Na+, K+, and Cl-, RAPID¬Point 500e results were also compared to the central chemistry analyzer AU5822 (Beckman Coulter, Brea, CA, USA). RESULTS: The coefficients of variation of within-laboratory imprecision for all analytes were less than 5%, within the range of acceptable criteria. The linearity values for all analytes were confirmed within manufacturer claimed range. RAPIDPoint 500e correlated well with both RAPIDPoint 500 and AU5822. Correlation coefficients were over 0.95 for all analytes. CONCLUSIONS: The analytical performance of RAPIDPoint 500e was clinically acceptable.

Analytical Performance Evaluation of Goldstream Fungus (1-3)-ß-D Glucan.

BACKGROUND: (1-3)-ß-D-glucan (BDG) is a fast and simple assay to diagnose invasive fungal infection. In this study, we evaluated the performance of the Goldstream BDG assay (Beijing Gold Mountainriver Tech Development) performed on the automated analyzer, IGL-200 (Genobio Pharmaceutical). METHODS: The precision and linearity of the Goldstream BDG assay were evaluated according to Clinical and Laboratory Standards Institute procedures. BDG results performed on the IGL-200 were compared to a manual photometer, MB-80A (Genobio Pharmaceutical). The manufacturer-provided reference interval was verified. RESULTS: Within-laboratory imprecision (% Coefficient of Variation) was 9.4%. The best polynomial fit was third-order within the manufacturer's claimed linear range (32.0 - 830.0 pg/mL). The BDG assay performed on IGL-200 and MB-80A showed a total agreement of 97.6%. All healthy subjects were within range of the manufacture provided reference interval. CONCLUSIONS: The analytical performance of the Goldstream BDG assay was clinically acceptable.

Novel interaction between neurotrophic factor-α1/carboxypeptidase E and serotonin receptor, 5-HTR1E, protects human neurons against oxidative/neuroexcitotoxic stress via ß-arrestin/ERK signaling.

Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H2O2-induced oxidative stress. Cell survival assay in ß-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was ß-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited ß-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of ß-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the ß-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection.

Unmet needs in palliative care for patients with common non-cancer diseases: a cross-sectional study.

BACKGROUND: Non-cancer patients experience the chronic process of disease that increases the patients' suffering as well as families' care burden. Although two-thirds of deaths are caused by non-cancer diseases, there is a lack of studies on palliative care for non-cancer patients. This study identified the palliative care needs and satisfaction, anxiety and depression, and health-related quality of life (HRQOL) of non-cancer patients and identified the factors influencing their HRQOL. METHODS: A cross-sectional survey design was employed. Participants were 114 non-cancer patients with chronic heart failure, stroke, end-stage renal disease, or end-stage liver disease who were admitted to the general ward of a tertiary hospital in South Korea. Measures included the Palliative Care Needs and Satisfaction Scale, the Hospital Anxiety and Depression Scale, and the Medical Outcome Study 36-items Short Form Health Survey version 2. Data were analysed with descriptive statistics, independent t-tests, analyses of variance, Pearson's correlations, and multiple linear regression analyses. RESULTS: The average score of palliative care needs was 3.66 ± 0.62, which falls between 'moderate' and 'necessary'. Among the four domains, the average score of palliative care needs in the psychosocial domain was the highest: 3.83 ± 0.67. Anxiety was nearly in the normal range (7.48 ± 3.60; normal range = 0-7) but depression was higher than normal (9.17 ± 3.71; normal range = 0-7). Similar to patients with cancer, physical HRQOL (38.89 ± 8.69) and mental HRQOL (40.43 ± 11.19) were about 80% of the general population's score (50 points). Duration of disease and physical performance were significant factors associated with physical HRQOL, whereas physical performance, anxiety, and depression were significant factors associated with mental HRQOL. CONCLUSION: It is necessary to maintain non-cancer patients' physical performance and assess and manage their mental health in advance for effective palliative care. This study provides relevant information that can be used to develop a tailored palliative care model for non-cancer patients.

Assay Sensitivity Difference Can Induce Anti-Hepatitis A Virus IgM Non-Reactive But Total (IgM and IgG) Reactive Results in Early Acute Hepatitis A.

Although anti-hepatitis A virus (HAV) IgM non-reactive and anti-HAV total (immunoglobulin [Ig] M and IgG) reactive results are generally interpreted as immunity to HAV, some early acute hepatitis A patients show the same results. We compared IgM detection sensitivity between anti-HAV IgM and anti-HAV total assays. Acute hepatitis A patients' samples were serially diluted and tested with Elecsys anti-HAV IgM and total assay (Roche Diagnostics). This resulted in anti-HAV IgM non-reactive but anti-HAV total reactive results. Samples of two hepatitis A patients showing false-negative anti-HAV IgM at initial presentation were analyzed with Elecsys, Atellica (Siemens Healthineers), and Alinity (Abbott Laboratories) HAV assays. Elecsys, Atellica, and Alinity anti-HAV IgM converted reactive on hospital day 3, whereas Elecsys and Atellica anti-HAV total results were reactive from hospital day 1. The anti-HAV total assay had higher sensitivity in detecting IgM antibodies than the anti-HAV IgM assay.

How Do We Start Palliative Care for Patients With End-Stage Liver Disease?

Patients with end-stage liver disease undergo repetitive patterns of recovery and deterioration and are burdened with uncertainty. Although quality of life is low in patients with end-stage liver disease and their family members, few studies have been conducted to identify what palliative care should be provided for them. This integrative review aimed to explore palliative care for patients with end-stage liver disease, focusing on the components and outcome measurements for further research. After searching for studies on palliative care for end-stage liver disease published between 1995 and 2017, 12 studies that met the inclusion criteria were analyzed. The common components of palliative care for patients with liver disease were: (a) an interdisciplinary approach, (b) early palliative care, (c) discussion goals of care with patient and family members, (d) symptom management, and (e) psychosocial support. It was reported that patients who were provided palliative care had improved itching, well-being, appetite, anxiety, fatigue, and depression, increased the number of do-not-resuscitate orders, palliative care consultations, and decreased length of stay. These findings could guide the development of palliative care for end-stage liver disease patients.

Association of Smoking Media Literacy with Vietnamese and South Korean Adolescents' Susceptibility to Smoking.

Studies on how smoking media literacy (SML) is associated with susceptibility to smoking among adolescents in South Korea and Vietnam are scarce. Thus, we examined the association of SML with susceptibility to smoking among adolescents in these countries to initiate a collaborative global health program. In total, 460 adolescents (Vietnam: 277, South Korea: 183) aged 15-18 completed an online cross-sectional survey. SML was measured using the 15-item SML scale. Susceptibility to smoking was measured by three questions on future smoking and if offered a cigarette by a friend. A multiple logistic regression model explored the association of SML with susceptibility to smoking. The study revealed that higher SML was significantly associated with lower susceptibility to smoking among Vietnamese, but not South Korean adolescents. Further studies to identify pathways between other factors associated with SML and susceptibility to smoking are needed to develop culture-specific intervention strategies for smoking prevention.

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.