Abstract

The angiotensin II receptors AT1R and AT2R serve as key components of the renin–angiotensin–aldosterone system. AT1R has a central role in the regulation of blood pressure, but the function of AT2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT2R bound to an AT2R-selective ligand and to an AT1R/AT2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or β-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure–activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.

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Acknowledgements

This work was supported by the National Institutes of Health (NIH) grants R01 GM108635 (V.C.) and U54 GM094618 (V.K., V.C. and R.C.S.); the National Science Foundation (NSF) grant 1231306 (U.W. and W.L.); the Helmholtz Association through program oriented funds (T.A.W. and A.T.). A.T. acknowledges financial support from ‘X-probe’ funded by the European Union’s 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement 637295. Parts of this research were carried out at the Coherent X-ray Imaging (CXI) end station of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, operated by Stanford University on behalf of the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515, and at the GM/CA CAT and IMCA-CAT of the Advanced Photon Source, Argonne National Laboratory. Parts of the sample delivery system used at LCLS for this research was funded by the NIH grant P41GM103393. Computational part of the study was supported by the University of Southern California Center for High-Performance Computing and Communications (https://hpcc.usc.edu/). We thank J. Velasquez for help with molecular biology, M. Chu for help with baculovirus expression, M. Hanson for help with crystallographic data processing and A. Walker for assistance with manuscript preparation.

Author information

Affiliations

  1. Department of Chemistry, Bridge Institute, University of Southern California, Los Angeles, California 90089, USA

    • Haitao Zhang
    • , Gye Won Han
    • , Andrii Ishchenko
    • , Kate L. White
    • , Anastasiia Sadybekov
    • , Raymond C. Stevens
    • , Vsevolod Katritch
    •  & Vadim Cherezov
  2. Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, China

    • Haitao Zhang
  3. Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, USA

    • Alexander Batyuk
    •  & Mark S. Hunter
  4. Department of Biological Sciences, Bridge Institute, University of Southern California, Los Angeles, California 90089, USA

    • Kate L. White
    • , Nilkanth Patel
    • , Raymond C. Stevens
    •  & Vsevolod Katritch
  5. MRL, Merck & Co., Inc., 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USA

    • Beata Zamlynny
    • , Margarita Garcia-Calvo
    •  & Payal Sheth
  6. MRL, Merck & Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, USA

    • Michael T. Rudd
    • , Kaspar Hollenstein
    • , Kerim Babaoglu
    • , Eric L. Moore
    •  & Stephen M. Soisson
  7. Center for Free Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany

    • Alexandra Tolstikova
    •  & Thomas A. White
  8. Department of Physics, University of Hamburg, Luruper Chaussee 149, 22761 Hamburg, Germany

    • Alexandra Tolstikova
  9. Department of Physics, Arizona State University, Tempe, Arizona 85287, USA

    • Uwe Weierstall
  10. School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA

    • Wei Liu
  11. MRL, Merck & Co., Inc., 503 Louise Lane, North Wales, Pennsylvania 19454, USA

    • Ryan D. Katz
    • , Jennifer M. Shipman
    •  & Sujata Sharma

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Contributions

K.H., S.M.S., R.C.S., V.K. and V.C. conceived and managed the project. H.Z. designed, optimized, purified, and characterized receptor constructs for structural studies, crystallized the receptor in LCP. H.Z. and A.I. collected and processed synchrotron data. H.Z., A.B., A.I., M.S.H., U.W., W.L. and V.C. collected XFEL data. A.B., A.T. and T.A.W. processed XFEL data. G.W.H., H.Z. and A.B. solved and refined the structures. M.T.R., K.H., K.B., E.L.M., S.M.S. and S.S. interpreted the structure and designed experiments. R.D.K. and J.M.S. prepared VLPs for binding studies. P.S., M.G.-C. and B.Z. designed the binding experiments. B.Z. carried out radioligand-binding assays with VLPs. H.Z., B.Z., M.G.-C., A.S., N.P. and P.S. analysed the data and compiled the figures for the manuscript. N.P., A.S. and V.K. performed docking and molecular dynamics simulations. K.L.W. performed radioligand-binding experiments with receptor mutants. M.T.R., K.H. and K.B. selected compounds for SAR study and interpreted the data. H.Z., V.K. and V.C. wrote the manuscript with contributions from M.T.R. and K.H.

Competing interests

B.Z., M.T.R., K.H., K.B., E.L.M., S.M.S., R.D.K., J.M.S., S.S., M.G.-C. and P.S. are employees of Merck & Co., Inc., Kenilworth, New Jersey, USA, receive salary and research support from the company and may own stock and/or stock options in the company. Other authors declare no competing financial interests.

Corresponding authors

Correspondence to Vsevolod Katritch or Vadim Cherezov.

Reviewer Information Nature thanks R. M. Carey, A. Hallberg and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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