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Crystal structure of pre-activated arrestin p44

Nature volume 497, pages 142–146 (2013)Cite this article

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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.

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Protein Data Bank

Data deposits

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

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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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Authors and Affiliations

  1. Institut für Medizinische Physik und Biophysik (CC2), Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany,

    Yong Ju Kim, Klaus Peter Hofmann, Hui-Woog Choe & Martha E. Sommer

  2. Zentrum für Biophysik und Bioinformatik, Humboldt-Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany,

    Klaus Peter Hofmann

  3. Departments of Biochemistry and Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, M5S 1A8, Ontario, Canada

    Oliver P. Ernst

  4. Institut für Medizinische Physik und Biophysik (CC2), AG Protein X-ray Crystallography, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany,

    Patrick Scheerer

  5. Department of Chemistry, College of Natural Science, Chonbuk National University, Chonju, 561-756, South Korea

    Hui-Woog Choe

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Contributions

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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