Abstract
G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.
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References
Pierce, K.L., Premont, R.T. & Lefkowitz, R.J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650 (2002).
Lefkowitz, R.J. A brief history of G-protein coupled receptors (Nobel Lecture). Angew. Chem. Int. Edn. Engl. 52, 6366–6378 (2013).
Lagerström, M.C. & Schiöth, H.B. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat. Rev. Drug Discov. 7, 339–357 (2008).
Kobilka, B.K. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal. Biochem. 231, 269–271 (1995).
Rajagopal, S., Rajagopal, K. & Lefkowitz, R.J. Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat. Rev. Drug Discov. 9, 373–386 (2010).
Violin, J.D., Crombie, A.L., Soergel, D.G. & Lark, M.W. Biased ligands at G-protein-coupled receptors: promise and progress. Trends Pharmacol. Sci. 35, 308–316 (2014).
Soergel, D.G., Subach, R.A., Cowan, C.L., Violin, J.D. & Lark, M.W. First clinical experience with TRV027: pharmacokinetics and pharmacodynamics in healthy volunteers. J. Clin. Pharmacol. 53, 892–899 (2013).
Soergel, D.G. et al. Biased agonism of the μ-opioid receptor by TRV130 increases analgesia and reduces on-target adverse effects versus morphine: a randomized, double-blind, placebo-controlled, crossover study in healthy volunteers. Pain 155, 1829–1835 (2014).
Kahsai, A.W. et al. Multiple ligand-specific conformations of the β2-adrenergic receptor. Nat. Chem. Biol. 7, 692–700 (2011).
Liu, J.J., Horst, R., Katritch, V., Stevens, R.C. & Wüthrich, K. Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335, 1106–1110 (2012).
Manglik, A. et al. Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell 161, 1101–1111 (2015).
Rasmussen, S.G. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011).
Rasmussen, S.G. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011).
Granier, S. & Kobilka, B. A new era of GPCR structural and chemical biology. Nat. Chem. Biol. 8, 670–673 (2012).
Ghosh, E., Kumari, P., Jaiman, D. & Shukla, A.K. Methodological advances: the unsung heroes of the GPCR structural revolution. Nat. Rev. Mol. Cell Biol. 16, 69–81 (2015).
Kobilka, B. The structural basis of G-protein-coupled receptor signaling (Nobel Lecture). Angew. Chem. Int. Edn. Engl. 52, 6380–6388 (2013).
Weichert, D. et al. Covalent agonists for studying G protein-coupled receptor activation. Proc. Natl. Acad. Sci. USA 111, 10744–10748 (2014).
Rosenbaum, D.M. et al. Structure and function of an irreversible agonist-β(2) adrenoceptor complex. Nature 469, 236–240 (2011).
Que-Gewirth, N.S. & Sullenger, B.A. Gene therapy progress and prospects: RNA aptamers. Gene Ther. 14, 283–291 (2007).
Ng, E.W. et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat. Rev. Drug Discov. 5, 123–132 (2006).
Rusconi, C.P. et al. RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 419, 90–94 (2002).
Lincoff, A.M. et al. REGULATE-PCI Investigators. Effect of the REG1 anticoagulation system versus bivalirudin on outcomes after percutaneous coronary intervention (REGULATE-PCI): a randomised clinical trial. Lancet 387, 349–356 (2016).
Ratner, M. Next-generation AMD drugs to wed blockbusters. Nat. Biotechnol. 32, 701–702 (2014).
Zhou, J. et al. Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible cells and inhibits HIV-1 infectivity. Chem. Biol. 22, 379–390 (2015).
Daniels, D.A., Sohal, A.K., Rees, S. & Grisshammer, R. Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin, NTS-1. Anal. Biochem. 305, 214–226 (2002).
Lee, G. et al. RNA based antagonist of NMDA receptors. ACS Chem. Neurosci. 5, 559–567 (2014).
Pratico, E.D., Sullenger, B.A. & Nair, S.K. Identification and characterization of an agonistic aptamer against the T cell costimulatory receptor, OX40. Nucleic Acid Ther. 23, 35–43 (2013).
Vinkenborg, J.L., Karnowski, N. & Famulok, M. Aptamers for allosteric regulation. Nat. Chem. Biol. 7, 519–527 (2011).
Tesmer, J.J. Crystallographic pursuit of a protein-RNA aptamer complex. Methods Mol. Biol. 1380, 151–160 (2016).
Oberthür, D. et al. Crystal structure of a mirror-image L-RNA aptamer (Spiegelmer) in complex with the natural L-protein target CCL2. Nat. Commun. 6, 6923 (2015).
Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).
Ellington, A.D. & Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).
Ozer, A., Pagano, J.M. & Lis, J.T. New technologies provide quantum changes in the scale, speed, and success of SELEX methods and aptamer characterization. Mol. Ther. Nucleic Acids 3, e183 (2014).
Shendure, J. & Ji, H. Next-generation DNA sequencing. Nat. Biotechnol. 26, 1135–1145 (2008).
Chae, P.S. et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat. Methods 7, 1003–1008 (2010).
Wang, J. et al. Synthesis of β2-AR agonist BI-167107. Youji Huaxue 33, 634–639 (2013).
Whorton, M.R. et al. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc. Natl. Acad. Sci. USA 104, 7682–7687 (2007).
Weiss, D.R. et al. Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor. ACS Chem. Biol. 8, 1018–1026 (2013).
Wisler, J.W. et al. A unique mechanism of beta-blocker action: carvedilol stimulates beta-arrestin signaling. Proc. Natl. Acad. Sci. USA 104, 16657–16662 (2007).
De Lean, A., Stadel, J.M. & Lefkowitz, R.J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J. Biol. Chem. 255, 7108–7117 (1980).
Hoffman, B.B. & Lefkowitz, R.J. Adrenergic receptors in the heart. Annu. Rev. Physiol. 44, 475–484 (1982).
Staus, D.P. et al. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol. Pharmacol. 85, 472–481 (2014).
Jiang, J. et al. The architecture of Tetrahymena telomerase holoenzyme. Nature 496, 187–192 (2013).
Peisley, A. & Skiniotis, G. 2D projection analysis of GPCR complexes by negative stain electron microscopy. Methods Mol. Biol. 1335, 29–38 (2015).
Kahsai, A.W., Rajagopal, S., Sun, J. & Xiao, K. Monitoring protein conformational changes and dynamics using stable-isotope labeling and mass spectrometry. Nat. Protoc. 9, 1301–1319 (2014).
Steyaert, J. & Kobilka, B.K. Nanobody stabilization of G protein-coupled receptor conformational states. Curr. Opin. Struct. Biol. 21, 567–572 (2011).
Hino, T. et al. G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody. Nature 482, 237–240 (2012).
Adams, J.J. & Sidhu, S.S. Synthetic antibody technologies. Curr. Opin. Struct. Biol. 24, 1–9 (2014).
Ivetac, A. & McCammon, J.A. Mapping the druggable allosteric space of G-protein coupled receptors: a fragment-based molecular dynamics approach. Chem. Biol. Drug Des. 76, 201–217 (2010).
Wootten, D., Christopoulos, A. & Sexton, P.M. Emerging paradigms in GPCR allostery: implications for drug discovery. Nat. Rev. Drug Discov. 12, 630–644 (2013).
Denisov, I.G., Grinkova, Y.V., Lazarides, A.A. & Sligar, S.G. Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J. Am. Chem. Soc. 126, 3477–3487 (2004).
Bompiani, K.M., Monroe, D.M., Church, F.C. & Sullenger, B.A. A high affinity, antidote-controllable prothrombin and thrombin-binding RNA aptamer inhibits thrombin generation and thrombin activity. J. Thromb. Haemost. 10, 870–880 (2012).
Shenoy, S.K. et al. beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor. J. Biol. Chem. 281, 1261–1273 (2006).
Williams, R.J. & Kelly, E. Measurement of adenylyl cyclase activity in cell membranes. Methods Mol. Biol. 41, 63–77 (1995).
Acknowledgements
R.J.L. is an investigator with the Howard Hughes Medical Institute (HHMI). This work was supported in part by grants from the US National Institutes of Health to R.J.L. (HL16037) and to B.A.S. (R01HL65222). We gratefully acknowledge B. Kobilka (Stanford University, Stanford, CA) and G. Skiniotis (University of Michigan, Ann Arbor, Michigan) for stimulating ideas and helpful discussions; we are also grateful to S. Rajagopal and J.C. Snyder of Duke University for discussions and critical reading of the manuscript; we thank O. Fedrigo and N. Hoang at the Genome Sequencing and Analysis Core Resource (Duke University) for library preparation support and quality control analysis and for performing next-generation DNA sequencing; we also acknowledge the use of transmission electron microscopy at the Shared Materials Instrumentation Facility (Duke University). T.J.C. is supported by an NHLBI grant of the National Institutes of Health (F30HL129803). We also thank X. Chen (Changzhou University, Jiangsu, China) for the supply of BI167107; A.K. Shukla (IIT, Kanpur, India), E. Pratico (Duke University), J. Kim (Duke University), and K. Xiao (University of Pittsburg) for valuable assistance with reagents; X. Jiang and W. Capel for excellent technical assistance; and D. Addison and Q. Lennon for secretarial assistance.
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A.W.K., J.W.W., S.A., B.A.S., and R.J.L. conceived the study. A.W.K., J.W.W., K.M.B., and H.D. performed selection and enrichment analysis. A.W.K., J.W.W., L.M.W., B.A.S., and R.J.L. designed NGS strategies. A.W.K., J.L., and H.D. performed Illumina-NGS library construction, preparation, and quality control analysis. A.W.K., J.L., H.D., X.Q., and L.M.W. participated in writing custom scripts and NGS data analysis. A.W.K. and J.L. performed aptamer synthesis, biotinylation, and fluorescence studies. S.M.D., K.M.A., and S.M.A. conducted BLI experiments. A.W.K., J.W.W., J.L., D.P.S., B.P., A.R.B.T., H.D., and R.T.S. participated in binding studies, receptor functionality tests, and reconstitution in HDL particles. S.A. conducted functional experiments. T.J.C. and A.R.B.T., with assistance from A.W.K. and J.L., performed EM imaging and particle analysis. A.W.K., B.A.S., and R.J.L. wrote the manuscript. All authors contributed to the preparation and editing of the manuscript.
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Kahsai, A., Wisler, J., Lee, J. et al. Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor. Nat Chem Biol 12, 709–716 (2016). https://doi.org/10.1038/nchembio.2126
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DOI: https://doi.org/10.1038/nchembio.2126
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