Structure of a nanobody-stabilized active state of the β2 adrenoceptor (original) (raw)

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Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for β2AR–Nb80 are deposited in the Protein Data Bank (accession code 3P0G).

References

1. Rosenbaum, D. M., Rasmussen, S. G. & Kobilka, B. K. The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009)
   Article ADS CAS Google Scholar
2. Scheerer, P. et al. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008)
   Article ADS CAS Google Scholar
3. Park, J. H., Scheerer, P., Hofmann, K. P., Choe, H. W. & Ernst, O. P. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008)
   Article ADS CAS Google Scholar
4. Li, J., Edwards, P. C., Burghammer, M., Villa, C. & Schertler, G. F. Structure of bovine rhodopsin in a trigonal crystal form. J. Mol. Biol. 343, 1409–1438 (2004)
   Article CAS Google Scholar
5. Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science [see comments]. 289, 739–745 (2000)
   Article ADS CAS Google Scholar
6. Vogel, R. & Siebert, F. Conformations of the active and inactive states of opsin. J. Biol. Chem. 276, 38487–38493 (2001)
   Article CAS Google Scholar
7. Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into β-adrenergic receptor function. Science 318, 1266–1273 (2007)
   Article ADS CAS Google Scholar
8. Rasmussen, S. G. et al. Crystal structure of the human β2 adrenergic G-protein-coupled receptor. Nature 450, 383–387 (2007)
   Article ADS CAS Google Scholar
9. Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007)
   Article ADS CAS Google Scholar
10. Hanson, M. A. et al. A specific cholesterol binding site is established by the 2.8 Å structure of the human β2-adrenergic receptor. Structure 16, 897–905 (2008)
    Article CAS Google Scholar
11. Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008)
    Article ADS CAS Google Scholar
12. Jaakola, V. P. et al. The 2.6 Angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322, 1211–1217 (2008)
    Article ADS CAS Google Scholar
13. Ghanouni, P. et al. Functionally different agonists induce distinct conformations in the G protein coupling domain of the β2 adrenergic receptor. J. Biol. Chem. 276, 24433–24436 (2001)
    Article CAS Google Scholar
14. Rosenbaum, D. M. et al. Structure and function of an irreversible agonist–β2 adrenoceptor complex. Nature doi:10.1038/nature09665 (this issue).
15. Yao, X. J. et al. The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex. Proc. Natl Acad. Sci. USA 106, 9501–9506 (2009)
    Article ADS CAS Google Scholar
16. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448 (1993)
    Article ADS CAS Google Scholar
17. Ballesteros, J. A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein coupled receptors. Meth. Neurosci. 25, 366–428 (1995)
    Article CAS Google Scholar
18. Strader, C. D. et al. Identification of residues required for ligand binding to the β-adrenergic receptor. Proc. Natl Acad. Sci. USA 84, 4384–4388 (1987)
    Article ADS CAS Google Scholar
19. Liapakis, G. et al. The forgotten serine. A critical role for Ser-2035.42 in ligand binding to and activation of the β2-adrenergic receptor. J. Biol. Chem. 275, 37779–37788 (2000)
    Article CAS Google Scholar
20. Wieland, K., Zuurmond, H. M., Krasel, C., Ijzerman, A. P. & Lohse, M. J. Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor. Proc. Natl Acad. Sci. USA 93, 9276–9281 (1996)
    Article ADS CAS Google Scholar
21. Shi, L. et al. β2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch. J. Biol. Chem. 277, 40989–40996 (2002)
    Article CAS Google Scholar
22. Ahuja, S. & Smith, S. O. Multiple switches in G protein-coupled receptor activation. Trends Pharmacol. Sci. 30, 494–502 (2009)
    Article CAS Google Scholar
23. Pellissier, L. P. et al. Conformational toggle switches implicated in basal constitutive and agonist-induced activated states of 5-hydroxytryptamine-4 receptors. Mol. Pharmacol. 75, 982–990 (2009)
    Article CAS Google Scholar
24. Altenbach, C., Kusnetzow, A. K., Ernst, O. P., Hofmann, K. P. & Hubbell, W. L. High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation. Proc. Natl Acad. Sci. USA 105, 7439–7444 (2008)
    Article ADS CAS Google Scholar
25. Parnot, C., Miserey-Lenkei, S., Bardin, S., Corvol, P. & Clauser, E. Lessons from constitutively active mutants of G protein-coupled receptors. Trends Endocrinol. Metab. 13, 336–343 (2002)
    Article CAS Google Scholar
26. 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)
    Article CAS Google Scholar
27. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nature Protocols 4, 706–731 (2009)
    Article CAS Google Scholar
28. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)
    Article CAS Google Scholar
29. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)
    Article CAS Google Scholar
30. Afonine, P. V., Grosse-Kunstleve, R. W. & Adams, P. D. A robust bulk-solvent correction and anisotropic scaling procedure. Acta Crystallogr. D 61, 850–855 (2005)
    Article Google Scholar
31. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT . Acta Crystallogr. D 60, 2210–2221 (2004)
    Article CAS Google Scholar
32. 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)
    Article ADS CAS Google Scholar

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Acknowledgements

We acknowledge support from National Institutes of Health Grants NS028471 and GM083118 (B.K.K.), GM56169 (W.I.W.), P01 GM75913 (S.H.G), and P60DK-20572 (R.K.S.), the Mathers Foundation (B.K.K. and W.I.W.), the Lundbeck Foundation (Junior Group Leader Fellowship, S.G.F.R.), the University of Michigan Biomedical Sciences Scholars Program (R.K.S.), the Fund for Scientific Research of Flanders (FWO-Vlaanderen) and the Institute for the encouragement of Scientific Research and Innovation of Brussels (ISRIB) (E.P. and J.S.).

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Author notes

1. Søren G. F. Rasmussen, Hee-Jung Choi and Juan Jose Fung: These authors contributed equally to this work.

Authors and Affiliations

1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, 94305, California, USA
   Søren G. F. Rasmussen, Hee-Jung Choi, Juan Jose Fung, Daniel M. Rosenbaum, Foon Sun Thian, Tong Sun Kobilka, William I. Weis & Brian K. Kobilka
2. Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark,
   Søren G. F. Rasmussen
3. Department of Structural Biology, Stanford University School of Medicine, 299 Campus Drive, Stanford, 94305, California, USA
   Hee-Jung Choi & William I. Weis
4. Department of Molecular and Cellular Interactions, Vlaams Instituut voor Biotechnologie (VIB), Vrije Universiteit Brussel, B-1050 Brussels, Belgium,
   Els Pardon & Jan Steyaert
5. Structural Biology Brussels, Vrije Universiteit Brussel, B-1050 Brussels, Belgium ,
   Els Pardon & Jan Steyaert
6. Boehringer Ingelheim Pharma GmbH & Co. KG, Germany
   Paola Casarosa, Andreas Schnapp, Ingo Konetzki & Alexander Pautsch
7. Department of Chemistry, University of Wisconsin, Madison, 53706, Wisconsin, USA
   Pil Seok Chae & Samuel H. Gellman
8. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, 48109, Michigan, USA
   Brian T. DeVree & Roger K. Sunahara

Authors

1. Søren G. F. Rasmussen
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2. Hee-Jung Choi
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3. Juan Jose Fung
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4. Els Pardon
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5. Paola Casarosa
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6. Pil Seok Chae
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7. Brian T. DeVree
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8. Daniel M. Rosenbaum
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9. Foon Sun Thian
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10. Tong Sun Kobilka
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11. Andreas Schnapp
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12. Ingo Konetzki
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13. Roger K. Sunahara
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14. Samuel H. Gellman
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15. Alexander Pautsch
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16. Jan Steyaert
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17. William I. Weis
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18. Brian K. Kobilka
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Contributions

S.G.F.R. screened and characterized high affinity agonists, identified and determined dissociation rate of BI-167107, screened, identified and characterized MNG-3, performed selection and characterization of nanobodies, purified and crystallized the receptor with Nb80 in LCP, optimized crystallization conditions, grew crystals for data collection, reconstituted receptor in HDL particles and determined the effect of Nb80 and Gs on receptor conformation and ligand binding affinities, assisted with data collection and preparing the manuscript. H.-J.C. processed diffraction data, solved and refined the structure, and assisted with preparing the manuscript. J.J.F. expressed, purified, selected and characterized nanobodies, purified and crystallized receptor with nanobodies in bicelles, assisted with growing crystals in LCP, and assisted with data collection. E.P. performed immunization, cloned and expressed nanobodies, and performed the initial selections. J.S. supervised nanobody production. P.S.K. and S.H.G. provided MNG-3 detergent for stabilization of purified β2AR. B.T.D. and R.K.S. provided ApoA1 and Gs protein, and reconstituted β2AR in HDL particles with Gs. D.M.R. characterized the usefulness of MNG-3 for crystallization in LCP and assisted with manuscript preparation. F.S.T. expressed β2AR in insect cells and with T.S.K performed the initial stage of β2AR purification. A.P., A.S. assisted in selection of the high-affinity agonist BI-167107. I.K. synthesized BI-167,107. P.C. characterized the functional properties of BI-167,107 in CHO cells. W.I.W. oversaw data processing, structure determination and refinement, and assisted with writing the manuscript. B.K.K. was responsible for the overall project strategy and management, prepared β2AR in lipid vesicles for immunization, harvested and collected data on crystals, and wrote the manuscript.

Corresponding authors

Correspondence toWilliam I. Weis or Brian K. Kobilka.

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The authors declare no competing financial interests.

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Rasmussen, S., Choi, HJ., Fung, J. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor.Nature 469, 175–180 (2011). https://doi.org/10.1038/nature09648

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• Received: 06 July 2010
• Accepted: 01 November 2010
• Published: 12 January 2011
• Issue Date: 13 January 2011
• DOI: https://doi.org/10.1038/nature09648