Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse ...physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with at least six cognate G protein-coupled receptors. Herein, we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analyses. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.
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•LPA1 structure determined with a selective antagonist•Structure-based design led to antagonists with improved characteristics•The LPA1 binding pocket permits extracellular ligand access unlike related S1P1R•Structural analysis predicts metabolic products of cannabinoid ligands bind to LPA1
Structural analyses of a human lysophosphatidic acid receptor reveal plasticity in ligand recognition and suggest mechanisms for modulation of receptors with distinct functions by common ligand scaffolds.
Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of a fusion partner that was inserted into the third intracellular loop. Using chimeras ...of the human β2-adrenergic and human A2A adenosine receptors, we present the methodology and data for the initial selection of an expanded set of fusion partners for crystallizing GPCRs. In particular, use of the thermostabilized apocytochrome b562RIL as a fusion partner displays certain advantages over previously utilized fusion proteins, resulting in a significant improvement in stability and structure of GPCR-fusion constructs.
► A method was developed for the selection of fusion domains for GPCR crystallization ► Apocytochrome b562RIL has advantages over previously utilized T4 lysozyme ► Diffraction quality crystals of two engineered GPCRs were successfully grown ► The method led to the crystal structure of the A2A adenosine receptor at 1.8 Å
ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans. Here we ...compare four x-ray structures of the bacterial ABC lipid flippase, MsbA, trapped in different conformations, two nucleotide-bound structures and two in the absence of nucleotide. Comparison of the nucleotide-free conformations of MsbA reveals a flexible hinge formed by extracellular loops 2 and 3. This hinge allows the nucleotide-binding domains to disassociate while the ATP-binding half sites remain facing each other. The binding of the nucleotide causes a packing rearrangement of the transmembrane helices and changes the accessibility of the transporter from cytoplasmic (inward) facing to extracellular (outward) facing. The inward and outward openings are mediated by two different sets of transmembrane helix interactions. Altogether, the conformational changes between these structures suggest that large ranges of motion may be required for substrate transport.
Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR ...structures. We reengineered the human A 2A adenosine receptor by replacing its third intracellular loop with apocytochrome b⁵⁶² RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp 2.50 . Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.
Crystal Structure of a Lipid G Protein—Coupled Receptor Hanson, Michael A.; Roth, Christopher B.; Jo, Euijung ...
Science (American Association for the Advancement of Science),
02/2012, Letnik:
335, Številka:
6070
Journal Article
Recenzirano
Odprti dostop
The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein—coupled ...sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P₁-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P₁, resulting in the modulation of immune and stremal cell responses.
Tirzepatide substantially reduced hemoglobin A1c (HbA1c) and body weight in subjects with type 2 diabetes (T2D) compared with the glucagon-like peptide 1 receptor agonist dulaglutide. Improved ...glycemic control was associated with lower circulating triglycerides and lipoprotein markers and improved markers of beta-cell function and insulin resistance (IR), effects only partially attributable to weight loss.
Assess plasma metabolome changes mediated by tirzepatide.
Phase 2b trial participants were randomly assigned to receive weekly subcutaneous tirzepatide, dulaglutide, or placebo for 26 weeks. Post hoc exploratory metabolomics and lipidomics analyses were performed.
Post hoc analysis.
259 subjects with T2D.
Tirzepatide (1, 5, 10, 15 mg), dulaglutide (1.5 mg), or placebo.
Changes in metabolite levels in response to tirzepatide were assessed against baseline levels, dulaglutide, and placebo using multiplicity correction.
At 26 weeks, a higher dose tirzepatide modulated a cluster of metabolites and lipids associated with IR, obesity, and future T2D risk. Branched-chain amino acids, direct catabolic products glutamate, 3-hydroxyisobutyrate, branched-chain ketoacids, and indirect byproducts such as 2-hydroxybutyrate decreased compared to baseline and placebo. Changes were significantly larger with tirzepatide compared with dulaglutide and directly proportional to reductions of HbA1c, homeostatic model assessment 2-IR indices, and proinsulin levels. Proportional to metabolite changes, triglycerides and diglycerides were lowered significantly compared to baseline, dulaglutide, and placebo, with a bias toward shorter and highly saturated species.
Tirzepatide reduces body weight and improves glycemic control and uniquely modulates metabolites associated with T2D risk and metabolic dysregulation in a direction consistent with improved metabolic health.
Multidrug resistance (MDR) is a serious medical problem and presents a major challenge to the treatment of disease and the development of novel therapeutics. ABC transporters that are associated with ...multidrug resistance (MDRABC transporters) translocate hydrophobic drugs and lipids from the inner to the outer leaflet of the cell membrane. To better elucidate the structural basis for the "flip-flop" mechanism of substrate movement across the lipid bilayer, we have determined the structure of the lipid flippase MsbA from Escherichia coli by x-ray crystallography to a resolution of 4.5 angstroms. MsbA is organized as a homodimer with each subunit containing six transmembrane α-helices and a nucleotide-binding domain. The asymmetric distribution of charged residues lining a central chamber suggests a general mechanism for the translocation of substrate by MsbA and other MDR-ABC transporters. The structure of MsbA can serve as a model for the MDR-ABC transporters that confer multidrug resistance to cancer cells and infectious microorganisms.
The dual burden of enteric infection and childhood malnutrition continues to be a global health concern and a leading cause of morbidity and death among children. Campylobacter infection, in ...particular, is highly prevalent in low- and middle-income countries, including Bangladesh. We examined longitudinal data to evaluate the trajectories of change in child growth, and to identify associations with Campylobacter infection and household factors. The study analyzed data from 265 children participating in the MAL-ED Study in Mirpur, Bangladesh. We applied latent growth curve modelling to evaluate the trajectories of change in children's height, as measured by length-for-age z-score (LAZ), from age 0-24 months. Asymptomatic and symptomatic Campylobacter infections were included as 3- and 6-month lagged time-varying covariates, while household risk factors were included as time-invariant covariates. Maternal height and birth order were positively associated with LAZ at birth. An inverse association was found between increasing age and LAZ. Campylobacter infection prevalence increased with age, with over 70% of children 18-24 months of age testing positive for infection. In the final model, Campylobacter infection in the preceding 3-month interval was negatively associated with LAZ at 12, 15, and 18 months of age; similarly, infection in the preceding 6-month interval was negatively associated with LAZ at 15, 18, and 21 months of age. Duration of antibiotic use and access to treated drinking water were negatively associated with Campylobacter infection, with the strength of the latter effect increasing with children's age. Campylobacter infection had a significant negative effect on child's growth and this effect was most powerful between 12 and 21 months. The treatment of drinking water and increased antibiotic use have a positive indirect effect on linear child growth trajectory, acting via their association with Campylobacter infection.
The role of cholesterol in eukaryotic membrane protein function has been attributed primarily to an influence on membrane fluidity and curvature. We present the 2.8 Å resolution crystal structure of ...a thermally stabilized human β2-adrenergic receptor bound to cholesterol and the partial inverse agonist timolol. The receptors pack as monomers in an antiparallel association with two distinct cholesterol molecules bound per receptor, but not in the packing interface, thereby indicating a structurally relevant cholesterol-binding site between helices I, II, III, and IV. Thermal stability analysis using isothermal denaturation confirms that a cholesterol analog significantly enhances the stability of the receptor. A consensus motif is defined that predicts cholesterol binding for 44% of human class A receptors, suggesting that specific sterol binding is important to the structure and stability of other G protein-coupled receptors, and that this site may provide a target for therapeutic discovery.
Purpose
To identify and evaluate methods for assessing pediatric patient-reported outcome (PRO) data quality at the individual level.
Methods
We conducted a systematic literature review to identify ...methods for detecting invalid responses to PRO measures. Eight data quality indicators were applied to child-report data collected from 1780 children ages 8–11 years. We grouped children with similar data quality patterns and tested for between-group differences in factors hypothesized to influence self-report capacity.
Results
We identified 126 articles that described 494 instances in which special measures or statistical techniques were applied to evaluate data quality at the individual level. We identified 22 data quality indicator subtypes: 9 direct methods (require administration of special items) and 13 archival techniques (statistical procedures applied to PRO data post hoc). Application of archival techniques to child-report PRO data revealed 3 distinct patterns (or classes) of the data quality indicators. Compared to class 1 (56%), classes 2 (36%) and 3 (8%) had greater variation in their PRO item responses. Three archival indicators were especially useful for differentiating plausible item response variation (class 2) from statistically unlikely response patterns (class 3). Neurodevelopmental conditions, which are associated with a range of cognitive processing challenges, were more common among children in class 3.
Conclusion
A multi-indicator approach is needed to identify invalid PRO responses. Once identified, assessment environments and measurement tools should be adapted to best support these individuals’ self-report capacity. Individual-level data quality indicators can be used to gauge the effectiveness of these accommodations.
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