scholarly journals Biophysical and functional characterization of Norrin signaling through Frizzled4

2018 ◽  
Vol 115 (35) ◽  
pp. 8787-8792 ◽  
Author(s):  
Injin Bang ◽  
Hee Ryung Kim ◽  
Andrew H. Beaven ◽  
Jinuk Kim ◽  
Seung-Bum Ko ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Wnt Signaling ◽  
Conformational Changes ◽  
Cellular Response ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Intracellular Loop ◽  
Ligand Interaction ◽  
Rich Domain ◽  
Linker Domain

Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate β-catenin–dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4CRD–Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4CRD to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced β-catenin–dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.

scholarly journals Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Lucas F. Ribeiro ◽  
Vanesa Amarelle ◽  
Liliane F. C. Ribeiro ◽  
María-Eugenia Guazzaroni
Keyword(s):  
Protein Engineering ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Protein Function ◽  
Cellular Response ◽  
Protein Domain ◽  
Signal Molecule ◽  
Synthetic Protein ◽  
Periplasmic Binding Proteins ◽  
Domain Insertion

All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an “off” to an “on” state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.

scholarly journals High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors

1999 ◽  
Vol 4 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Ilona Kariv ◽  
Michelle E. Stevens ◽  
Davette L. Behrens ◽  
Kevin R. Oldenburg
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Transmembrane Domain ◽  
Receptor Function ◽  
Camp Production ◽  
Ligand Interaction ◽  
G Protein Coupled ◽  
Native Ligand

Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.

scholarly journals CXCR3 Requires Tyrosine Sulfation for Ligand Binding and a Second Extracellular Loop Arginine Residue for Ligand-Induced Chemotaxis

2006 ◽  
Vol 26 (15) ◽  
pp. 5838-5849 ◽  
Author(s):  
Richard A. Colvin ◽  
Gabriele S. V. Campanella ◽  
Lindsay A. Manice ◽  
Andrew D. Luster
Keyword(s):  
Ligand Binding ◽  
Transmembrane Domain ◽  
Nk Cell ◽  
Cell Trafficking ◽  
Extracellular Loop ◽  
Ligand Interaction ◽  
Lower Affinity ◽  
Extracellular Domains ◽  
N Terminus ◽  
Charged Residues

ABSTRACT CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.

scholarly journals Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Open Biology ◽  
2021 ◽  
Vol 11 (4) ◽  
Author(s):  
Evelyn Ploetz ◽  
Gea K. Schuurman-Wolters ◽  
Niels Zijlstra ◽  
Amarins W. Jager ◽  
Douglas A. Griffith ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Transmembrane Domain ◽  
Substrate Binding ◽  
Titration Calorimetry ◽  
Uptake System ◽  
Conformational States ◽  
Binding Domains

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.

scholarly journals Functional dissection of the N-terminal extracellular domains of Frizzled 6 reveals their roles for receptor localization and Dishevelled recruitment

2018 ◽  
Vol 293 (46) ◽  
pp. 17875-17887 ◽  
Author(s):  
Jana Valnohova ◽  
Maria Kowalski-Jahn ◽  
Roger K. Sunahara ◽  
Gunnar Schulte
Keyword(s):  
Transmembrane Domain ◽  
Surface Expression ◽  
Receptor Activation ◽  
Membrane Expression ◽  
Rich Domain ◽  
Receptor Localization ◽  
Evolutionarily Conserved ◽  
Activation Mechanisms ◽  
Receptor Surface Expression ◽  
Linker Domain

The Frizzled (FZD) proteins belong to class F of G protein–coupled receptors (GPCRs) and are essential for various pathways involving the secreted lipoglycoproteins of the wingless/int-1 (WNT) family. A WNT-binding cysteine-rich domain (CRD) in FZDs is N-terminally located and connected to the seven transmembrane domain–spanning receptor core by a linker domain that has a variable length in different FZD homologs. However, the function and importance of this linker domain are poorly understood. Here we used systematic mutagenesis of FZD6 to define the minimal N-terminal domain sufficient for receptor surface expression and recruitment of the intracellular scaffold protein Dishevelled (DVL). Further, we identified a triad of evolutionarily conserved cysteines in the FZD linker domain that is crucial for receptor membrane expression and recruitment of DVL. Our results are in agreement with the concept that the conserved cysteines in the linker domain of FZDs assist with the formation of a common secondary structure in this region. We propose that this structure could be involved in agonist binding and receptor activation mechanisms that are similar to the binding and activation mechanisms known for other GPCRs.

scholarly journals Ligand-Induced Conformational Dynamics of A Tyramine Receptor from Sitophilus oryzae

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mac Kevin E. Braza ◽  
Jerrica Dominique N. Gazmen ◽  
Eizadora T. Yu ◽  
Ricky B. Nellas
Keyword(s):  
Conformational Changes ◽  
Cellular Response ◽  
Conformational Dynamics ◽  
Sitophilus Oryzae ◽  
Md Simulations ◽  
Homology Model ◽  
Activation Process ◽  
Intracellular Loop ◽  
Binding Effect ◽  
Natural Ligand

Abstract Tyramine receptor (TyrR) is a biogenic amine G protein-coupled receptor (GPCR) associated with many important physiological functions in insect locomotion, reproduction, and pheromone response. Binding of specific ligands to the TyrR triggers conformational changes, relays the signal to G proteins, and initiates an appropriate cellular response. Here, we monitor the binding effect of agonist compounds, tyramine and amitraz, to a Sitophilus oryzae tyramine receptor (SoTyrR) homology model and their elicited conformational changes. All-atom molecular dynamics (MD) simulations of SoTyrR-ligand complexes have shown varying dynamic behavior, especially at the intracellular loop 3 (IL3) region. Moreover, in contrast to SoTyrR-tyramine, SoTyrR-amitraz and non-liganded SoTyrR shows greater flexibility at IL3 residues and were found to be coupled to the most dominant motion in the receptor. Our results suggest that the conformational changes induced by amitraz are different from the natural ligand tyramine, albeit being both agonists of SoTyrR. This is the first attempt to understand the biophysical implication of amitraz and tyramine binding to the intracellular domains of TyrR. Our data may provide insights into the early effects of ligand binding to the activation process of SoTyrR.

scholarly journals Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

2015 ◽  
Vol 89 (13) ◽  
pp. 6633-6645 ◽  
Author(s):  
Kristen M. Ogden ◽  
Liya Hu ◽  
Babal K. Jha ◽  
Banumathi Sankaran ◽  
Susan R. Weiss ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Cellular Response ◽  
Substrate Binding ◽  
Core Structure ◽  
Structural Basis ◽  
Rnase L ◽  
Binding Mechanism ◽  
Oligoadenylate Synthetase ◽  
Terminal Domain

ABSTRACTSynthesis of 2′-5′-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined HΦ(S/T)Φ (where Φ is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave β-sheet that contains the catalytic cleft, but it lacks two α-helical regions and two β-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A.IMPORTANCEThe C-terminal domain (CTD) of rotavirus VP3 is a 2H phosphoesterase that cleaves 2′-5′-oligoadenylates (2-5A), potent activators of an important innate cellular antiviral pathway. 2H phosphoesterase superfamily proteins contain two conserved catalytic motifs and a proposed core structure. Here, we present structures of a viral 2H phosphoesterase, the rotavirus VP3 CTD, alone and in complex with its substrate, 2-5A. The domain lacks two α-helical regions and β-strands present in other 2H phosphoesterases. A loop of the protein undergoes significant structural changes upon substrate binding. Together with our bioinformatics and biochemical findings, the crystal structures suggest that the RVA VP3 CTD domain is a streamlined version of a cellular enzyme that shares a ligand-binding mechanism with other 2H phosphodiesterases that cleave 2-5A but differs from those of 2H phosphodiesterases that cleave other substrates. These findings may aid in the future design of antivirals targeting viral phosphodiesterases with cleavage specificity for 2-5A.

scholarly journals Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain

2000 ◽  
Vol 352 (2) ◽  
pp. 363-372 ◽  
Author(s):  
Andor KISS ◽  
Kalthoum FARAH ◽  
Joon KIM ◽  
Robert J. GARRIOCK ◽  
Thomas A. DRYSDALE ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Phosphorylation Site ◽  
Functional Expression ◽  
Amino Acid Identity ◽  
Nucleoside Transporters ◽  
Intracellular Loop ◽  
Equilibrative Nucleoside Transporters

Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human>mouse>rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1has 88% and 78% amino acid identity with rat ENT1 and human ENT1 respectively; mENT2 is more highly conserved, with 94% and 88% identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids (designated mENT1b) that adds a potential protein kinase CK2 phosphorylation site in the central intracellular loop of the transporter, and is similar, in this regard, to the human and rat ENT1 orthologues. The other cDNA has a 5′-untranslated region sequence that is distinct from that of full-length mENT1. Microinjection of mENT1, mENT1b or mENT2 cRNA into Xenopus oocytes resulted in enhanced uptake of [3H]uridine by the oocytes relative to that seen in water-injected controls. mENT1-mediated, but not mENT2-mediated, [3H]uridine uptake was inhibited by nitrobenzylthioinosine and dilazep. Dipyridamole inhibited both mENT1 and mENT2, but was significantly more effective against mENT1. Adenosine inhibited both systems with a similar potency, as did a range of other purine and pyrimidine nucleosides. These results are compatible with the known characteristics of the native mENT1 and mENT2 transporters.

scholarly journals Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

2020 ◽  
Author(s):  
Evelyn Ploetz ◽  
Gea K. Schuurman-Wolters ◽  
Niels Zijlstra ◽  
Amarins W. Jager ◽  
Douglas A. Griffith ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Substrate Binding ◽  
List Type ◽  
Binding Affinities ◽  
Proof Of Concept ◽  
Conformational States ◽  
Binding Domains

ABSTRACTThe ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid, and asparagine in Gram-positive bacteria. It features two extracytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from L. lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster-resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously.HIGHLIGHTSResolved crystal structure of tandem SBD1-2 of GlnPQ from Lactococcus lactisConformational states and ligand binding affinities of individual domains SBD1 and SBD2 are similar to tandem SBD1-2No cooperative effects are seen for different ligands for SBDs in the tandemProof of concept experiments show that PIFE-FRET can monitor SBD conformations and protein-protein interaction simultaneously

scholarly journals Structures of metabotropic GABAB receptor

2020 ◽  
Author(s):  
Makaía M. Papasergi-Scott ◽  
Michael J. Robertson ◽  
Alpay B. Seven ◽  
Ouliana Panova ◽  
Jesper M. Mathiesen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Cellular Response ◽  
Transmembrane Domain ◽  
Gabab Receptor ◽  
Rational Drug Design ◽  
Protein Activation ◽  
Brain Physiology ◽  
Rational Drug ◽  
G Protein Activation

AbstractGABA (γ-aminobutyric acid) stimulation of the metabotropic GABAB receptor results in prolonged inhibition of neurotransmission that is central to brain physiology. GABAB belongs to the Family C of G protein-coupled receptors (GPCRs), which operate as dimers to relay synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins. GABAB, however, is unique in its function as an obligate heterodimer in which agonist binding and G protein activation take place on distinct subunits. Here we show structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signaling assays and atomistic simulations, the structures reveal an essential role for the GABAB extracellular loop 2 (ECL2) in relaying structural transitions by ordering the linker connecting the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of both GABAB subunits caps and interacts with the hydrophilic head of a phospholipid occupying the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G protein. These results provide a starting framework to decipher mechanistic modes of signal transduction mediated by GABAB dimers and have important implications for rational drug design targeting these receptors.

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