The Experts below are selected from a list of 6198 Experts worldwide ranked by ideXlab platform
Serge Muyldermans - One of the best experts on this subject based on the ideXlab platform.
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Applications of Nanobodies.
Annual review of animal biosciences, 2020Co-Authors: Serge MuyldermansAbstract:Unique, functional, homodimeric heavy chain-only antibodies, devoid of light chains, are circulating in the blood of Camelidae. These antibodies recognize their cognate antigen via one single domain, known as VHH or Nanobody. This serendipitous discovery made three decades ago has stimulated a growing number of researchers to generate highly specific Nanobodies against a myriad of targets. The small size, strict monomeric state, robustness, and easy tailoring of these Nanobodies have inspired many groups to design innovative Nanobody-based multi-domain constructs to explore novel applications. As such, Nanobodies have been employed as an exquisite research tool in structural, cell, and developmental biology. Furthermore, Nanobodies have demonstrated their benefit for more sensitive diagnostic tests. Finally, several Nanobody-based constructs have been designed to develop new therapeutic products. Expected final online publication date for the Annual Review of Animal Biosciences, Volume 9 is February 16, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Site-Selective Functionalization of Nanobodies Using Intein-Mediated Protein Ligation for Innovative Bioconjugation
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Geert-jan Graulus, Duy Tien Ta, Rebekka Hansen, Brecht Billen, Erik Royackers, Nick Devoogdt, Jean-paul Noben, Serge Muyldermans, Huong Tran, Wanda GuedensAbstract:An expression strategy is presented in order to produce nanobodies modified with a clickable alkyne functionality at their C-terminus via the intein-mediated protein ligation (IPL) technique. The protocol focuses on the cytoplasmic expression and extraction of a Nanobody-intein-chitin binding domain (CBD) fusion protein in E. coli SHuffle® T7 cells, in the commonly used Luria-Bertani (LB) medium. The combination of these factors results in a high yield and nearly complete alkynation of the Nanobody at its C-terminus via IPL. The resulting alkynated nanobodies retain excellent binding capacity toward the Nanobody targeted antigen. The presented protocol benefits from time- and cost-effectiveness and allows for a feasible upscaling of functionalized (here alkynated) nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to (1) novel biosurface applications that demand for homogeneously oriented nanobodies having their active site fully accessible for target (e.g., biomarker) binding, and (2) innovative applications such as localized drug delivery and image guided surgery by covalent "click" chemistry coupling of these alkynated nanobodies to a multitude of azide-containing counterparts as there are drug containing polymers and contrast labeling agents.
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The structural basis of Nanobody unfolding reversibility and thermoresistance
Scientific Reports, 2018Co-Authors: Patrick Kunz, Katinka Zinner, Tanja Bartoschik, Norbert Mücke, Serge Muyldermans, Jorg D HoheiselAbstract:Nanobodies represent the variable binding domain of camelid heavy-chain antibodies and are employed in a rapidly growing range of applications in biotechnology and biomedicine. Their success is based on unique properties including their reported ability to reversibly refold after heat-induced denaturation. This view, however, is contrasted by studies which involve irreversibly aggregating nanobodies, asking for a quantitative analysis that clearly defines Nanobody thermoresistance and reveals the determinants of unfolding reversibility and aggregation propensity. By characterizing nearly 70 nanobodies, we show that irreversible aggregation does occur upon heat denaturation for the large majority of binders, potentially affecting application-relevant parameters like stability and immunogenicity. However, by deriving aggregation propensities from apparent melting temperatures, we show that an optional disulfide bond suppresses Nanobody aggregation. This effect is further enhanced by increasing the length of a complementarity determining loop which, although expected to destabilize, contributes to Nanobody stability. The effect of such variations depends on environmental conditions, however. Nanobodies with two disulfide bonds, for example, are prone to lose their functionality in the cytosol. Our study suggests strategies to engineer nanobodies that exhibit optimal performance parameters and gives insights into general mechanisms which evolved to prevent protein aggregation.
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Exploiting sequence and stability information for directing Nanobody stability engineering.
Biochimica et biophysica acta. General subjects, 2017Co-Authors: Patrick Kunz, Serge Muyldermans, Cecile Vincke, Tilman Flock, Nicolas Soler, Moritz Zaiss, Yann G.-j. Sterckx, Damjana Kastelic, Jorg D HoheiselAbstract:Abstract Background Variable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics and therapy, engineering their stability is of particular interest. One important aspect is the improvement of thermostability, because it can have immediate effects on conformational stability, protease resistance and aggregation propensity of the protein. Methods We analyzed the sequences and thermostabilities of 78 purified Nanobody binders. From this data, potentially stabilizing amino acid variations were identified and studied experimentally. Results Some mutations improved the stability of nanobodies by up to 6.1 °C, with an average of 2.3 °C across eight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stability and aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some instances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasons for this contradiction between prediction and experiment were investigated. Conclusions The results reveal a mutational strategy to improve the biophysical behavior of Nanobody binders and indicate a species-specificity of Nanobody architecture. General significance This study illustrates the potential and limitations of engineering Nanobody thermostability by merging sequence information with stability data, an aspect that is becoming increasingly important with the recent development of high-throughput biophysical methods.
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Generation and characterization of nanobodies targeting PSMA for molecular imaging of prostate cancer
Contrast media & molecular imaging, 2014Co-Authors: Mehdi Evazalipour, Tony Lahoutte, Kobra Omidfar, Matthias D'huyvetter, Bahram Soltani Tehrani, Mohsen Abolhassani, Shahriyar Abdoli, Roghaye Arezumand, Hamid Morovvati, Serge MuyldermansAbstract:Nanobodies show attractive characteristics for tumor targeting in cancer diagnosis and therapy. A radiolabeled Nanobody binding the prostate-specific membrane antigen (PSMA) could offer a noninvasive strategy to select prostate cancer patients eligible for PSMA-targeted therapies. We here describe the generation, production and in vivo evaluation of anti-PSMA nanobodies. Nanobodies were derived from heavy-chain-only antibodies, raised in immunized dromedaries. Binding characteristics were evaluated through ELISA and flow cytometry. Selected nanobodies were radiolabeled with 99mTc at their hexahistidine tail, after which cell binding capacity and internalization were evaluated on PSMApos LNCaP and PSMAneg PC3 cell lines. In vivo tumor targeting was analyzed in both LNCaP and PC3 xenografted mice through SPECT/microCT and tissue sampling. A panel of 72 generated clones scored positive on ELISA, all contributing to three Nanobody groups, of which group 3 dominated with 70 clones. ELISA and FACS analysis led to the selection of two dominant nanobodies. 99mTc-labeled PSMA6 and PSMA30 both showed specific binding on LNCAP cells, but not on PC3 cells. 99mTc-PSMA30 internalized significantly more in LNCaP cells compared to 99mTc-PSMA6. Higher absolute tumor uptake and tumor-to-normal organ ratios were observed for 99mTc-PSMA30 compared with 99mTc-PSMA6 and a 99mTc-control Nanobody in LNCaP but not in PC3 tumor-bearing mice. PSMA30 Nanobody has improved targeting characteristics both in vitro as well as in vivo compared with PSMA6 and the control Nanobody, and was therefore selected as our in-house-developed lead compound for PSMA targeting. Copyright © 2014 John Wiley & Sons, Ltd.
Daniel Nietlispach - One of the best experts on this subject based on the ideXlab platform.
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Conformational plasticity of ligand-bound and ternary GPCR complexes studied by ^19F NMR of the β_1-adrenergic receptor
Nature Communications, 2020Co-Authors: J. Niclas Frei, Richard W. Broadhurst, Andrew J. Y. Jones, Florian Gabriel, Aditi Tandale, Binesh Shrestha, Mark J Bostock, Andras Solt, Daniel NietlispachAbstract:The β_1-adrenergic receptor (β_1AR) is a G-protein-coupled receptor (GPCRs) that binds catecholamine ligands. Here the authors employ site-specific labelling and ^19F NMR measurements to characterise the structural changes and dynamics in the cytoplasmic region of β_1AR upon agonist stimulation and coupling to a G_s-protein-mimetic Nanobody. G-protein-coupled receptors (GPCRs) are allosteric signaling proteins that transmit an extracellular stimulus across the cell membrane. Using ^19F NMR and site-specific labelling, we investigate the response of the cytoplasmic region of transmembrane helices 6 and 7 of the β_1-adrenergic receptor to agonist stimulation and coupling to a G_s-protein-mimetic Nanobody. Agonist binding shows the receptor in equilibrium between two inactive states and a pre-active form, increasingly populated with higher ligand efficacy. Nanobody coupling leads to a fully active ternary receptor complex present in amounts correlating directly with agonist efficacy, consistent with partial agonism. While for different agonists the helix 6 environment in the active-state ternary complexes resides in a well-defined conformation, showing little conformational mobility, the environment of the highly conserved NPxxY motif on helix 7 remains dynamic adopting diverse, agonist-specific conformations, implying a further role of this region in receptor function. An inactive Nanobody-coupled ternary receptor form is also observed.
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Insight into partial agonism by observing multiple equilibria for ligand-bound and G_s-mimetic Nanobody-bound β_1-adrenergic receptor
Nature Communications, 2017Co-Authors: Andras Solt, Binesh Shrestha, Mark J Bostock, Prashant Kumar, Tony Warne, Christopher G. Tate, Daniel NietlispachAbstract:β_1-adrenergic receptors are expressed in cardiac tissue and stimulated by the sympathetic nervous system. Here, the authors use NMR spectroscopy to unravel the conformational diversity upon β_1-adrenergic receptor activation and provide structural insights into partial agonism and basal activity. A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., G_s and β-arrestin. Using ^13C methyl methionine NMR for the β_1-adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with G_s-mimetic Nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free Nanobody–receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound Nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid Nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level.
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insight into partial agonism by observing multiple equilibria for ligand bound and gs mimetic Nanobody bound β1 adrenergic receptor
Nature Communications, 2017Co-Authors: Andras Solt, Binesh Shrestha, Mark J Bostock, Prashant Kumar, Tony Warne, Christopher G. Tate, Daniel NietlispachAbstract:A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., Gs and β-arrestin. Using 13C methyl methionine NMR for the β1-adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with Gs-mimetic Nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free Nanobody–receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound Nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid Nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level. β1-adrenergic receptors are expressed in cardiac tissue and stimulated by the sympathetic nervous system. Here, the authors use NMR spectroscopy to unravel the conformational diversity upon β1-adrenergic receptor activation and provide structural insights into partial agonism and basal activity.
Jan Steyaert - One of the best experts on this subject based on the ideXlab platform.
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An improved yeast surface display platform for the screening of Nanobody immune libraries.
Scientific reports, 2019Co-Authors: Tomasz Uchański, Els Pardon, Thomas Zögg, Jie Yin, Daopeng Yuan, Alexandre Wohlkonig, Baptiste Fischer, Daniel M. Rosenbaum, Brian K. Kobilka, Jan SteyaertAbstract:Fusions to the C-terminal end of the Aga2p mating adhesion of Saccharomyces cerevisiae have been used in many studies for the selection of affinity reagents by yeast display followed by flow cytometric analysis. Here we present an improved yeast display system for the screening of Nanobody immune libraries where we fused the Nanobody to the N-terminal end of Aga2p to avoid steric hindrance between the fused Nanobody and the antigen. Moreover, the display level of a cloned Nanobody on the surface of an individual yeast cell can be monitored through a covalent fluorophore that is attached in a single enzymatic step to an orthogonal acyl carrier protein (ACP). Additionally, the displayed Nanobody can be easily released from the yeast surface and immobilised on solid surfaces for rapid analysis. To prove the generic nature of this novel Nanobody discovery platform, we conveniently selected Nanobodies against three different antigens, including two membrane proteins.
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Nanobody enabled reverse pharmacology on g protein coupled receptors
Angewandte Chemie, 2018Co-Authors: Els Pardon, Cecilia Betti, Florent Chevillard, Karel Guillemyn, Peter Kolb, Steven Ballet, Toon Laeremans, Jan SteyaertAbstract:: The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a β2 -adrenoreceptor-Nanobody fusion locked in its active-state conformation by a G-protein-mimicking Nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to Nanobody-enabled reverse pharmacology.
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isolation of antigen binding camelid heavy chain antibody fragments nanobodies from an immune library displayed on the surface of pichia pastoris
Journal of Biotechnology, 2010Co-Authors: Stefan Ryckaert, Els Pardon, Jan Steyaert, Nico CallewaertAbstract:Yeast surface display is an efficient tool for isolating and engineering antibody fragments, both scFv and Fab. We describe the use of protein display on Pichia pastoris for the rapid selection of camelid antibodies composed only of heavy chains (nanobodies) from a library derived from a llama immunized with Green Fluorescent Protein. The library of Nanobody-coding sequences was fused to the C-terminal part of the Saccharomyces cerevisiae alpha-agglutinin gene (SAG1) and expressed in glycoengineered P. pastoris. A high efficiency transformation protocol yielded a library of 5x10(7) clones. About 80% of the clones strongly expressed the Nanobody fusion. Nanobody-displaying clones were rapidly enriched by fluorescence activated cell sorting (FACS), and GFP-specific Nanobody-displaying clones were isolated and equilibrium dissociation constants (K(d)) determined. This technology for displaying protein libraries on P. pastoris enables the isolation and engineering of antibody-derived molecules in a robust eukaryotic expression host.
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Nanobody aided structure determination of the epsi epsj pseudopilin heterodimer from vibrio vulnificus
Journal of Structural Biology, 2009Co-Authors: Els Pardon, Konstantin V Korotkov, Jan SteyaertAbstract:Pseudopilins form the central pseudopilus of the sophisticated bacterial type 2 secretion systems. The crystallization of the EpsI:EpsJ pseudopilin heterodimer from Vibrio vulnificus was greatly accelerated by the use of nanobodies, which are the smallest antigen-binding fragments derived from heavy-chain only camelid antibodies. Seven anti-EpsI:EpsJ nanobodies were generated and co-crystallization of EpsI:EpsJ Nanobody complexes yielded several crystal forms very rapidly. In the structure solved, the nanobodies are arranged in planes throughout the crystal lattice, linking layers of EpsI:EpsJ heterodimers. The EpsI:EpsJ dimer observed confirms a right-handed architecture of the pseudopilus, but, compared to a previous structure of the EpsI:EpsJ heterodimer, EpsI differs 6° in orientation with respect to EpsJ; one loop of EpsJ is shifted by ∼5 A due to interactions with the Nanobody; and a second loop of EpsJ underwent a major change of 17 A without contacts with the Nanobody. Clearly, nanobodies accelerate dramatically the crystallization of recalcitrant protein complexes and can reveal conformational flexibility not observed before.
Els Pardon - One of the best experts on this subject based on the ideXlab platform.
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An improved yeast surface display platform for the screening of Nanobody immune libraries.
Scientific reports, 2019Co-Authors: Tomasz Uchański, Els Pardon, Thomas Zögg, Jie Yin, Daopeng Yuan, Alexandre Wohlkonig, Baptiste Fischer, Daniel M. Rosenbaum, Brian K. Kobilka, Jan SteyaertAbstract:Fusions to the C-terminal end of the Aga2p mating adhesion of Saccharomyces cerevisiae have been used in many studies for the selection of affinity reagents by yeast display followed by flow cytometric analysis. Here we present an improved yeast display system for the screening of Nanobody immune libraries where we fused the Nanobody to the N-terminal end of Aga2p to avoid steric hindrance between the fused Nanobody and the antigen. Moreover, the display level of a cloned Nanobody on the surface of an individual yeast cell can be monitored through a covalent fluorophore that is attached in a single enzymatic step to an orthogonal acyl carrier protein (ACP). Additionally, the displayed Nanobody can be easily released from the yeast surface and immobilised on solid surfaces for rapid analysis. To prove the generic nature of this novel Nanobody discovery platform, we conveniently selected Nanobodies against three different antigens, including two membrane proteins.
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Nanobody enabled reverse pharmacology on g protein coupled receptors
Angewandte Chemie, 2018Co-Authors: Els Pardon, Cecilia Betti, Florent Chevillard, Karel Guillemyn, Peter Kolb, Steven Ballet, Toon Laeremans, Jan SteyaertAbstract:: The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a β2 -adrenoreceptor-Nanobody fusion locked in its active-state conformation by a G-protein-mimicking Nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to Nanobody-enabled reverse pharmacology.
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structures of p glycoprotein reveal its conformational flexibility and an epitope on the nucleotide binding domain
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Andrew B Ward, Els Pardon, Paul Szewczyk, Vinciane Grimard, Changwook Lee, Lorena Martinez, Rupak Doshi, Alexandra Caya, Mark Villaluz, Cristina CreggerAbstract:P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a Nanobody bound to the C-terminal side of the first nucleotide-binding domain. This Nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The Nanobody-bound structure also reveals a unique epitope on P-gp.
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structure of a Nanobody stabilized active state of the β2 adrenoceptor
Nature, 2011Co-Authors: Soren G F Rasmussen, Els Pardon, Daniel M. Rosenbaum, Heejung Choi, Juan Jose Fung, Paola Casarosa, Pil Seok Chae, Brian T Devree, Foon Sun Thian, Tong Sun KobilkaAbstract:G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (Nanobody) to the human b2 adrenergic receptor (b2AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-Nanobody complex. Comparison with the inactive b2AR structure reveals subtle changes in the binding
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isolation of antigen binding camelid heavy chain antibody fragments nanobodies from an immune library displayed on the surface of pichia pastoris
Journal of Biotechnology, 2010Co-Authors: Stefan Ryckaert, Els Pardon, Jan Steyaert, Nico CallewaertAbstract:Yeast surface display is an efficient tool for isolating and engineering antibody fragments, both scFv and Fab. We describe the use of protein display on Pichia pastoris for the rapid selection of camelid antibodies composed only of heavy chains (nanobodies) from a library derived from a llama immunized with Green Fluorescent Protein. The library of Nanobody-coding sequences was fused to the C-terminal part of the Saccharomyces cerevisiae alpha-agglutinin gene (SAG1) and expressed in glycoengineered P. pastoris. A high efficiency transformation protocol yielded a library of 5x10(7) clones. About 80% of the clones strongly expressed the Nanobody fusion. Nanobody-displaying clones were rapidly enriched by fluorescence activated cell sorting (FACS), and GFP-specific Nanobody-displaying clones were isolated and equilibrium dissociation constants (K(d)) determined. This technology for displaying protein libraries on P. pastoris enables the isolation and engineering of antibody-derived molecules in a robust eukaryotic expression host.
Andras Solt - One of the best experts on this subject based on the ideXlab platform.
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Conformational plasticity of ligand-bound and ternary GPCR complexes studied by ^19F NMR of the β_1-adrenergic receptor
Nature Communications, 2020Co-Authors: J. Niclas Frei, Richard W. Broadhurst, Andrew J. Y. Jones, Florian Gabriel, Aditi Tandale, Binesh Shrestha, Mark J Bostock, Andras Solt, Daniel NietlispachAbstract:The β_1-adrenergic receptor (β_1AR) is a G-protein-coupled receptor (GPCRs) that binds catecholamine ligands. Here the authors employ site-specific labelling and ^19F NMR measurements to characterise the structural changes and dynamics in the cytoplasmic region of β_1AR upon agonist stimulation and coupling to a G_s-protein-mimetic Nanobody. G-protein-coupled receptors (GPCRs) are allosteric signaling proteins that transmit an extracellular stimulus across the cell membrane. Using ^19F NMR and site-specific labelling, we investigate the response of the cytoplasmic region of transmembrane helices 6 and 7 of the β_1-adrenergic receptor to agonist stimulation and coupling to a G_s-protein-mimetic Nanobody. Agonist binding shows the receptor in equilibrium between two inactive states and a pre-active form, increasingly populated with higher ligand efficacy. Nanobody coupling leads to a fully active ternary receptor complex present in amounts correlating directly with agonist efficacy, consistent with partial agonism. While for different agonists the helix 6 environment in the active-state ternary complexes resides in a well-defined conformation, showing little conformational mobility, the environment of the highly conserved NPxxY motif on helix 7 remains dynamic adopting diverse, agonist-specific conformations, implying a further role of this region in receptor function. An inactive Nanobody-coupled ternary receptor form is also observed.
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Insight into partial agonism by observing multiple equilibria for ligand-bound and G_s-mimetic Nanobody-bound β_1-adrenergic receptor
Nature Communications, 2017Co-Authors: Andras Solt, Binesh Shrestha, Mark J Bostock, Prashant Kumar, Tony Warne, Christopher G. Tate, Daniel NietlispachAbstract:β_1-adrenergic receptors are expressed in cardiac tissue and stimulated by the sympathetic nervous system. Here, the authors use NMR spectroscopy to unravel the conformational diversity upon β_1-adrenergic receptor activation and provide structural insights into partial agonism and basal activity. A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., G_s and β-arrestin. Using ^13C methyl methionine NMR for the β_1-adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with G_s-mimetic Nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free Nanobody–receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound Nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid Nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level.
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insight into partial agonism by observing multiple equilibria for ligand bound and gs mimetic Nanobody bound β1 adrenergic receptor
Nature Communications, 2017Co-Authors: Andras Solt, Binesh Shrestha, Mark J Bostock, Prashant Kumar, Tony Warne, Christopher G. Tate, Daniel NietlispachAbstract:A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., Gs and β-arrestin. Using 13C methyl methionine NMR for the β1-adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with Gs-mimetic Nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free Nanobody–receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound Nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid Nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level. β1-adrenergic receptors are expressed in cardiac tissue and stimulated by the sympathetic nervous system. Here, the authors use NMR spectroscopy to unravel the conformational diversity upon β1-adrenergic receptor activation and provide structural insights into partial agonism and basal activity.
