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Crystal structure of pre-activated arrestin p44

Abstract

Arrestins interact with G-protein-coupled receptors (GPCRs) to block interaction with G proteins1,2 and initiate G-protein-independent signalling3. Arrestins have a bi-lobed structure that is stabilized by a long carboxy-terminal tail (C-tail), and displacement of the C-tail by receptor-attached phosphates activates arrestins for binding active GPCRs4. Structures of the inactive state of arrestin are available5,6, but it is not known how C-tail displacement activates arrestin for receptor coupling. Here we present a 3.0 Å crystal structure of the bovine arrestin-1 splice variant p44, in which the activation step is mimicked by C-tail truncation. The structure of this pre-activated arrestin is profoundly different from the basal state and gives insight into the activation mechanism. p44 displays breakage of the central polar core and other interlobe hydrogen-bond networks, leading to a 21° rotation of the two lobes as compared to basal arrestin-1. Rearrangements in key receptor-binding loops in the central crest region include the finger loop7,8,9, loop 139 (refs 8, 10, 11) and the sequence Asp 296–Asn 305 (or gate loop), here identified as controlling the polar core. We verified the role of these conformational alterations in arrestin activation and receptor binding by site-directed fluorescence spectroscopy. The data indicate a mechanism for arrestin activation in which C-tail displacement releases critical central-crest loops from restricted to extended receptor-interacting conformations. In parallel, increased flexibility between the two lobes facilitates a proper fitting of arrestin to the active receptor surface. Our results provide a snapshot of an arrestin ready to bind the active receptor, and give an insight into the role of naturally occurring truncated arrestins in the visual system.

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Figure 1: Structural differences between basal arrestin-1 and p44.
Figure 2: Comparison of loops that differ between basal arrestin-1 and p44.
Figure 3: Comparison of electrostatic surfaces of basal arrestin-1 and p44.
Figure 4: Rearrangement of interdomain hydrogen-bond networks in p44 and resulting interdomain rotation.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4J2Q.

References

  1. 1

    Wilden, U., Hall, S. W. & Kuhn, H. Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. Proc. Natl Acad. Sci. USA 83, 1174–1178 (1986)

    CAS  ADS  Article  Google Scholar 

  2. 2

    Lohse, M. J., Benovic, J. L., Codina, J., Caron, M. G. & Lefkowitz, R. J. β-Arrestin: a protein that regulates β-adrenergic receptor function. Science 248, 1547–1550 (1990)

    CAS  ADS  Article  Google Scholar 

  3. 3

    Shukla, A. K., Xiao, K. & Lefkowitz, R. J. Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling. Trends Biochem. Sci. 36, 457–469 (2011)

    CAS  Article  Google Scholar 

  4. 4

    Gurevich, V. V., Hanson, S. M., Song, X., Vishnivetskiy, S. A. & Gurevich, E. V. The functional cycle of visual arrestins in photoreceptor cells. Prog. Retin. Eye Res. 30, 405–430 (2011)

    CAS  Article  Google Scholar 

  5. 5

    Granzin, J. et al. X-ray crystal structure of arrestin from bovine rod outer segments. Nature 391, 918–921 (1998)

    CAS  ADS  Article  Google Scholar 

  6. 6

    Hirsch, J. A., Schubert, C., Gurevich, V. V. & Sigler, P. B. The 2.8 Å crystal structure of visual arrestin: a model for arrestin’s regulation. Cell 97, 257–269 (1999)

    CAS  Article  Google Scholar 

  7. 7

    Feuerstein, S. E. et al. Helix formation in arrestin accompanies recognition of photoactivated rhodopsin. Biochemistry 48, 10733–10742 (2009)

    CAS  Article  Google Scholar 

  8. 8

    Hanson, S. M. et al. Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin. Proc. Natl Acad. Sci. USA 103, 4900–4905 (2006)

    CAS  ADS  Article  Google Scholar 

  9. 9

    Sommer, M. E., Farrens, D. L., McDowell, J. H., Weber, L. A. & Smith, W. C. Dynamics of arrestin-rhodopsin interactions: loop movement is involved in arrestin activation and receptor binding. J. Biol. Chem. 282, 25560–25568 (2007)

    CAS  Article  Google Scholar 

  10. 10

    Kim, M. et al. Conformation of receptor-bound visual arrestin. Proc. Natl Acad. Sci. USA 109, 18407–18412 (2012)

    CAS  ADS  Article  Google Scholar 

  11. 11

    Vishnivetskiy, S. A., Baameur, F., Findley, K. R. & Gurevich, V. V. Critical role of central 139-loop in stability and binding selectivity of arrestin-1. J. Biol. Chem.. http://dx.doi.org/10.1074/jbc.M113.450031 jbc M113.4 50031 (2013)

  12. 12

    Gurevich, V. V. & Benovic, J. L. Visual arrestin interaction with rhodopsin. Sequential multisite binding ensures strict selectivity toward light-activated phosphorylated rhodopsin. J. Biol. Chem. 268, 11628–11638 (1993)

    CAS  PubMed  Google Scholar 

  13. 13

    Schröder, K., Pulvermüller, A. & Hofmann, K. P. Arrestin and its splice variant Arr1–370A (p44). Mechanism and biological role of their interaction with rhodopsin. J. Biol. Chem. 277, 43987–43996 (2002)

    Article  Google Scholar 

  14. 14

    Kirchberg, K. et al. Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process. Proc. Natl Acad. Sci. USA 108, 18690–18695 (2011)

    CAS  ADS  Article  Google Scholar 

  15. 15

    Schleicher, A., Kühn, H. & Hofmann, K. P. Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II. Biochemistry 28, 1770–1775 (1989)

    CAS  Article  Google Scholar 

  16. 16

    Smith, W. C. et al. A splice variant of arrestin. Molecular cloning and localization in bovine retina. J. Biol. Chem. 269, 15407–15410 (1994)

    CAS  PubMed  Google Scholar 

  17. 17

    Pulvermüller, A. et al. Functional differences in the interaction of arrestin and its splice variant, p44, with rhodopsin. Biochemistry 36, 9253–9260 (1997)

    Article  Google Scholar 

  18. 18

    Scheerer, P. et al. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008)

    CAS  ADS  Article  Google Scholar 

  19. 19

    Choe, H. W. et al. Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011)

    CAS  ADS  Article  Google Scholar 

  20. 20

    Gurevich, V. V. & Benovic, J. L. Visual arrestin binding to rhodopsin. Diverse functional roles of positively charged residues within the phosphorylation-recognition region of arrestin. J. Biol. Chem. 270, 6010–6016 (1995)

    CAS  Article  Google Scholar 

  21. 21

    Vishnivetskiy, S. A. et al. An additional phosphate-binding element in arrestin molecule. Implications for the mechanism of arrestin activation. J. Biol. Chem. 275, 41049–41057 (2000)

    CAS  Article  Google Scholar 

  22. 22

    Hanson, S. M. & Gurevich, V. V. The differential engagement of arrestin surface charges by the various functional forms of the receptor. J. Biol. Chem. 281, 3458–3462 (2006)

    CAS  Article  Google Scholar 

  23. 23

    Granzin, J. et al. Crystal structure of p44, a constitutively active splice variant of visual arrestin. J. Mol. Biol. 416, 611–618 (2012)

    CAS  Article  Google Scholar 

  24. 24

    Mansoor, S. E., Dewitt, M. A. & Farrens, D. L. Distance mapping in proteins using fluorescence spectroscopy: the tryptophan-induced quenching (TrIQ) method. Biochemistry 49, 9722–9731 (2010)

    CAS  Article  Google Scholar 

  25. 25

    Sommer, M. E., Hofmann, K. P. & Heck, M. Distinct loops in arrestin differentially regulate ligand binding within the GPCR opsin. Nature Commun. 3, 995 (2012)

    ADS  Article  Google Scholar 

  26. 26

    Sommer, M. E., Hofmann, K. P. & Heck, M. Arrestin-rhodopsin binding stoichiometry in isolated rod outer segment membranes depends on the percentage of activated receptors. J. Biol. Chem. 286, 7359–7369 (2011)

    CAS  Article  Google Scholar 

  27. 27

    Zhuang, T. et al. Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin. Proc. Natl Acad. Sci. USA 110, 942–947 (2012)

    ADS  Article  Google Scholar 

  28. 28

    Gurevich, V. V. et al. Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, β2-adrenergic, and m2 muscarinic cholinergic receptors. J. Biol. Chem. 270, 720–731 (1995)

    CAS  Article  Google Scholar 

  29. 29

    Azarian, S. M., King, A. J., Hallett, M. A. & Williams, D. S. Selective proteolysis of arrestin by calpain. Molecular characteristics and its effect on rhodopsin dephosphorylation. J. Biol. Chem. 270, 24375–24384 (1995)

    CAS  Article  Google Scholar 

  30. 30

    Sommer, M. E., Smith, W. C. & Farrens, D. L. Dynamics of arrestin-rhodopsin interactions: acidic phospholipids enable binding of arrestin to purified rhodopsin in detergent. J. Biol. Chem. 281, 9407–9417 (2006)

    CAS  Article  Google Scholar 

  31. 31

    Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)

    CAS  Article  Google Scholar 

  32. 32

    Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Article  Google Scholar 

  33. 33

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  34. 34

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  35. 35

    Vagin, A. A. et al. REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. D 60, 2184–2195 (2004)

    Article  Google Scholar 

  36. 36

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  Article  Google Scholar 

  37. 37

    Winn, M. D., Isupov, M. N. & Murshudov, G. N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D 57, 122–133 (2001)

    CAS  Article  Google Scholar 

  38. 38

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  Article  Google Scholar 

  39. 39

    Vaguine, A. A., Richelle, J. & Wodak, S. J. SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr. D 55, 191–205 (1999)

    CAS  Article  Google Scholar 

  40. 40

    Laskowski, R. A., Moss, D. S. & Thornton, J. M. Procheck: a program to check the stereo chemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)

    CAS  Article  Google Scholar 

  41. 41

    Hooft, R. W., Vriend, G., Sander, C. & Abola, E. E. Errors in protein structures. Nature 381, 272 (1996)

    CAS  ADS  Article  Google Scholar 

  42. 42

    McDonald, I. K. & Thornton, J. M. Satisfying hydrogen bonding potential in proteins. J. Mol. Biol. 238, 777–793 (1994)

    CAS  Article  Google Scholar 

  43. 43

    Wallace, A. C., Laskowski, R. A. & Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 8, 127–134 (1995)

    CAS  Article  Google Scholar 

  44. 44

    Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

    CAS  ADS  Article  Google Scholar 

  45. 45

    Poornam, G. P., Matsumoto, A., Ishida, H. & Hayward, S. A method for the analysis of domain movements in large biomolecular complexes. Proteins 76, 201–212 (2009)

    CAS  Article  Google Scholar 

  46. 46

    DeLano, W. L. The PyMOL Molecular Graphics System (DeLano Scientific, 2002)

  47. 47

    Sommer, M. E., Smith, W. C. & Farrens, D. L. Dynamics of arrestin-rhodopsin interactions: arrestin and retinal release are directly linked events. J. Biol. Chem. 280, 6861–6871 (2005)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank J. H. Park for help at the early stage of the project and B. Bauer, J. Engelmann, C. Koch and H. Seibel for technical assistance. We are grateful to U. Müller, M. Weiss and the scientific staff of the BESSY-MX/Helmholtz Zentrum Berlin für Materialien und Energie at beamlines BL 14.1, BL 14.2 and BL 14.3 operated by the Joint Berlin MX-Laboratory at the BESSY II electron storage ring (Berlin-Adlershof, Germany) and the scientific staff of the European Synchrotron Radiation Facility (ESRF, Grenoble) at beamlines ID14-1, ID 23-1, ID 23-2, ID 29S, ID 29 and ID 14-4 for continuous support. The data presented here were recorded at beamline ID 14-4 (ESRF, Grenoble). This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB449 to O.P.E., SFB740 to K.P.H. and O.P.E., SFB1078-B6 to P.S., SO1037/1-2 to M.E.S.), DFG Cluster of Excellence ‘Unifying Concepts in Catalysis’ (Research Field D3/E3-1 to P.S.), European Research Council (Advanced Investigator Grant (ERC-2009/249910-TUDOR to K.P.H.)), the Canada Excellence Research Chair program (to O.P.E.) and the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2012R1A1A2044752 to H.-W.C.). O.P.E. holds The Anne and Max Tanenbaum Chair in Neuroscience at the University of Toronto.

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K.P.H., O.P.E. and H.-W.C. designed the structural studies of p44. Y.J.K. performed p44 preparation, functional analysis and crystallization; Y.J.K., P.S. and H.-W.C. performed data collection and structural analysis; M.E.S. designed and performed functional assays and fluorescence measurements of labelled arrestin mutants; Y.J.K., K.P.H., P.S., H.-W.C. and M.E.S. analysed and interpreted data; M.E.S. wrote the paper with contributions from all co-authors.

Corresponding authors

Correspondence to Patrick Scheerer, Hui-Woog Choe or Martha E. Sommer.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-13, Supplementary Methods, Supplementary Tables 1-2, a Supplementary Discussion and Supplementary References. (PDF 7446 kb)

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Kim, Y., Hofmann, K., Ernst, O. et al. Crystal structure of pre-activated arrestin p44. Nature 497, 142–146 (2013). https://doi.org/10.1038/nature12133

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