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Crystal structures of the human adiponectin receptors

Nature volume 520, pages 312–316 (2015)Cite this article

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Abstract

Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases the activities of 5′ AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR), respectively, thereby contributing to healthy longevity as key anti-diabetic molecules. AdipoR1 and AdipoR2 were predicted to contain seven transmembrane helices with the opposite topology to G-protein-coupled receptors. Here we report the crystal structures of human AdipoR1 and AdipoR2 at 2.9 and 2.4 Å resolution, respectively, which represent a novel class of receptor structure. The seven-transmembrane helices, conformationally distinct from those of G-protein-coupled receptors, enclose a large cavity where three conserved histidine residues coordinate a zinc ion. The zinc-binding structure may have a role in the adiponectin-stimulated AMPK phosphorylation and UCP2 upregulation. Adiponectin may broadly interact with the extracellular face, rather than the carboxy-terminal tail, of the receptors. The present information will facilitate the understanding of novel structure–function relationships and the development and optimization of AdipoR agonists for the treatment of obesity-related diseases, such as type 2 diabetes.

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Figure 1: Overall structures of AdipoR1 and AdipoR2.
Figure 2: Sequence alignment of human AdipoR1 and AdipoR2.
Figure 3: The zinc-binding sites of AdipoR1 and AdipoR2.
Figure 4: The large internal cavities in the AdipoR1 and AdipoR2 structures.
Figure 5: The extracellular faces of AdipoR1 and AdipoR2.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors for the AdipoR1–Fv and AdipoR2–Fv structures have been deposited in the Protein Data Bank under the accession codes 3WXV and 3WXW, respectively.

Change history

  • 15 April 2015

    A minor editorial change was made to the legend of Figure 3 to recover the original label definition.

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Acknowledgements

We are grateful to the staffs of BL32XU at SPring-8 (proposals 2012A1332, 2012B1453, 2013A1008, 2013A1008, 2013B1034, 2013B1007, 2014A1007, 2014A1008 and 2014A1186), beamline I24 at Diamond Light Source, and beamline X06SA at the Swiss Light Source for their assistance in data collection. We thank R. Akasaka for protein analysis, M. Toyama, M. Inoue, M. Goto, M. Aoki and K. Ishii for expression plasmid preparation, M. Nishimoto, Y. Tomabechi and Y. Terazawa for technical assistance with protein expression and purification, and Y. Nishibaba, M. Yuasa and A. Hayashi for technical assistance and support with the activity assays of the mutants. This work was supported by grants from the Targeted Proteins Research Program (S.Y., T.K., S.I. and M.Y.), the Platform for Drug Discovery, Informatics and Structural Life Science (S.Y. and M.Y.), a Grant-in-Aid for Specially Promoted Research (26000012) (T.K.), Grants-in-Aid for Scientific Research (S) (20229008, 25221307) (T.K.), a Grant-in-Aid for Scientific Research (B) (26293216) (M.O.-I.), a Grant-in-Aid for Young Scientists (A) (30557236) (M. Iwabu), and the Translational Research Network Program (M.O.-I.), from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the research acceleration program of the Japan Science and Technology Agency (S.I.), and by the BBSRC (BB/G02325/1) (S.I.). The authors are grateful for the use of the Membrane Protein Laboratory funded by the Wellcome Trust (grant 062164/Z/00/Z) (S.I.) at the Diamond Light Source Limited.

Author information

Author notes

  1. Miki Okada-Iwabu, Masato Iwabu and Yoshihiro Nakamura: These authors contributed equally to this work.

Authors and Affiliations

  1. RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, 230-0045, Yokohama, Japan

    Hiroaki Tanabe, Yoshifumi Fujii, Yoshihiro Nakamura, Toshiaki Hosaka, Kanna Motoyama, Mariko Ikeda, Motoaki Wakiyama, Takaho Terada, Noboru Ohsawa, Masakatsu Hato, Takeshi Murata, So Iwata, Tomomi Kimura-Someya, Mikako Shirouzu & Shigeyuki Yokoyama

  2. Department of Biophysics and Biochemistry and Laboratory of Structural Biology, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Hiroaki Tanabe & Shigeyuki Yokoyama

  3. Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, 230-0045, Yokohama, Japan

    Hiroaki Tanabe, Yoshihiro Nakamura, Toshiaki Hosaka, Mariko Ikeda, Motoaki Wakiyama, Noboru Ohsawa, Masakatsu Hato, Tomomi Kimura-Someya & Mikako Shirouzu

  4. RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi-ku, 230-0045, Yokohama, Japan

    Hiroaki Tanabe, Yoshifumi Fujii, Yoshihiro Nakamura, Takaho Terada & Shigeyuki Yokoyama

  5. Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Miki Okada-Iwabu, Masato Iwabu, Toshimasa Yamauchi & Takashi Kadowaki

  6. Department of Integrated Molecular Science on Metabolic Diseases, 22nd Century Medical and Research Center, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Miki Okada-Iwabu, Masato Iwabu, Toshimasa Yamauchi & Takashi Kadowaki

  7. PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan

    Masato Iwabu

  8. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan

    Satoshi Ogasawara, Tomoya Hino, Takeshi Murata & So Iwata

  9. JST, Research Acceleration Program, Membrane Protein Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan

    Tomoya Hino, Takeshi Murata & So Iwata

  10. Department of Chemistry, Graduate School of Science, Chiba University, Yayoi-cho, Inage, Chiba, 263-8522, Japan

    Takeshi Murata

  11. Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London, SW7 2AZ, UK

    So Iwata

  12. Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, OX11 0DE, UK

    So Iwata

  13. RIKEN SPring-8 Center, Harima Institute, Kouto, Sayo, Hyogo, 679-5148, Japan

    So Iwata, Kunio Hirata, Yoshiaki Kawano & Masaki Yamamoto

  14. CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, 332-0012, Japan

    Toshimasa Yamauchi

Authors
  1. Hiroaki Tanabe
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  2. Yoshifumi Fujii
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  3. Miki Okada-Iwabu
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Contributions

H.T., T.K.-S., M.S., M.O.-I., M. Iwabu, T.Y., T.K. and S.Y. designed the research. H.T., K.M., M. Ikeda, M.W. and T.T. performed protein expression, purification and analyses of AdipoR1 and AdipoR2, while N.O. designed and constructed the expression plasmids. M.H. provided the lipidic mesophase crystallization techniques, and H.T. and K.M. performed the crystallization of the receptors. Y.F., H.T., Y.N. and T. Hosaka performed the X-ray diffraction data collection and the structural analysis. K.H., Y.K. and M.Y. optimized the microcrystal data collection strategy, using BL32XU at SPring-8. H.T. prepared the AdipoR1 and AdipoR2 immunogens, and H.T., K.M., S.O., T. Hino, T.M. and S.I. produced the anti-AdipoR1 monoclonal antibody. M.O.-I. and M. Iwabu assayed the activities of the mutants. H.T., T.K.-S., M.S., M.O.-I., M. Iwabu, T.Y., T.K. and S.Y. wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Toshimasa Yamauchi, Takashi Kadowaki or Shigeyuki Yokoyama.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Analysis of N-terminal deletion mutants of AdipoR1 and AdipoR2 and lattice packing of the AdipoR1–Fv and AdipoR2–Fv crystals.

a, Phosphorylation and amounts of AMPK in HEK293 cells transfected with full-length AdipoR1 (residues 1–375) or N-terminally truncated mutants (residues 47–375, 77–375, 89–375, 102–375 and 120–375), treated for 5 min with adiponectin (15 μg ml−1). b, UCP2 mRNA levels in HEK293 cells transfected with full-length AdipoR2 (residues 1–386) or N-terminally truncated mutants (residues 88–386 and 100–386), treated for 18 h with adiponectin (3 μg ml−1). All values are mean ± s.e.m. n = 3–4, three independent experiments. **P < 0.01 compared to control cells or as indicated (see Methods for statistical tests used). NS, not significant. c–h, Lattice packing of the AdipoR1–Fv crystals (c–e) and the AdipoR2–Fv crystals (f–h). AdipoR1, AdipoR2 and Fv are coloured green, cyan and grey, respectively. The AdipoR1–Fv and AdipoR2–Fv complexes crystallized with an anti-parallel arrangement of the receptor molecules.

Extended Data Figure 2 Comparison of the AdipoR1–Fv and AdipoR2–Fv structures.

a–c, Superimposition of the AdipoR1–Fv and AdipoR2–Fv complexes: side view (a), extracellular view (b), intracellular view (c). Fv was omitted from the intracellular view for clarity. d, Superimposition of the subdomains consisting of helices I, II, III and VII between AdipoR1 and AdipoR2. The Cα r.m.s.d. value is 0.34 Å. e, Helix 0 (pink) interacts hydrophobically with the cytoplasmic ends of helices I–III, and the ICL1 (purple), as represented by those of AdipoR1. In addition, the zinc ion firmly connects helices VII, II and III (Fig. 3). Therefore, helices VII, I, II and III (d) constitute a rigid subdomain in the 7TM-domain structures. By contrast, helices IV, V and VI are superimposed between AdipoR1 and AdipoR2 with a Cα r.m.s.d. value of 0.73 Å, and are likely to constitute the other subdomain, with some conformational differences in helix V between AdipoR1 and AdipoR2.

Extended Data Figure 3 Comparison of the AdipoR2 structure with other 7TM proteins.

The structures of 7TM proteins in the N-terminus-out topology were inverted and superimposed on the AdipoR2 structure in the C-terminus-out topology. Superimpositions of AdipoR2 (cyan) with the β2AR (PDB code 2RH1) (r.m.s.d. 3.9 Å) (a), the glucagon receptor (PDB code 4L6R) (r.m.s.d. 3.8 Å) (b), the metabotropic glutamate receptor 1 (PDB code 4OR2) (r.m.s.d. 2.9 Å) (c), the sphingosine 1-phosphate receptor 1 (PDB code 3V2Y)56 (r.m.s.d. 3.0 Å) (d), the A2A adenosine receptor (PDB code 2YDV)57 (r.m.s.d. 3.4 Å) (e), and sensory rhodopsin (PDB code 1XIO)58 (r.m.s.d. 3.1 Å) (f) in orange. The AdipoR2 and other 7TM protein structures are viewed parallel to the membrane (top) and from the extracellular and intracellular sides, respectively (bottom).

Extended Data Figure 4 The zinc-binding sites of AdipoR1 and AdipoR2.

a, b, The zinc-binding sites of AdipoR1 (a) and AdipoR2 (b) are conserved from mammals to plants. The conserved residues, the 3× His+Asp residues and a Ser residue, are shown in red. The side chains of Ser 187 (AdipoR1) and Ser 198 (AdipoR2) in helix II are located 3.7 and 3.8 Å, respectively, away from the zinc ion (data not shown and Fig. 3b). c, d, The amounts of AdipoR1 (c) and AdipoR2 (d) in HEK293 cells transfected with AdipoR1 (residues 89–375), AdipoR2 (residues 100–386) or a variety of mutants of AdipoR1 and AdipoR2 were analysed by western blotting, using an anti-Flag antibody. The label 89–375 indicates no mutation, and the other labels, such as His191Ala and 4Ala, indicate the single and multiple mutations (see text). The label 100–386 indicates no mutation, and the other labels, such as His202Ala and 4Ala, indicate the single and multiple mutations (see text). e, UCP2 mRNA levels in HEK293 cells transfected with full-length AdipoR2 (residues 1–386) or a zinc-binding site mutant. All values are presented as mean ± s.e.m. n = 3–4, three independent experiments, *P < 0.05, **P < 0.01 compared to control cells or as indicated (see Methods).

Extended Data Figure 5 The zinc-binding sites of soluble proteins.

a, b, The zinc-binding sites of Astacus astacus L. astacin (PDB code 1AST) (a) and human carbonic anhydrase II (PDB code 1CA2) (b). The zinc ion (magenta) is coordinated by three His residues and a water molecule (pink sphere).

Extended Data Figure 6 The cytoplasmic side of AdipoR1.

a, b, Structures of AdipoR1 residues 89–375 (a) and 120–375 (b) viewed parallel to the membrane. c, d, The cavity of AdipoR1 (residues 89–375 (c) and 120–375 (d)) viewed from the cytoplasmic side. Residues 120–375, including helix 0, the 7TM domain, and the CTR, are coloured green. The NTR (residues 89–119) is coloured orange.

Extended Data Figure 7 The cytoplasmic faces of AdipoR2 and AdipoR1.

a, b, Intracellular views of AdipoR1 (a) and AdipoR2 (b). The openings of the cavities are circled in red. The N-terminal regions of the AdipoRs are represented as surface models (orange). c, d, The ICL2 s of AdipoR1 (c) and AdipoR2 (d).

Extended Data Figure 8 Crystal packing of the CTRs of AdipoR1 and AdipoR2.

a, b, Crystal packing of the CTR of AdipoR1 with Fv (a) and the CTR of AdipoR2 with Fv (b). The CTR of AdipoR1 is tucked between the two Fv fragments, whereas the C-terminal tail of AdipoR2 contacts the framework region 1 of VH (orange). The CTRs are coloured purple.

Extended Data Figure 9 Expression of the AdipoR1 mutant proteins.

a, b, The amounts of AdipoR1 in HEK293 cells transfected with full-length AdipoR1 (residues 1–375) or a variety of mutants of AdipoR1 were analysed by western blotting, using an anti-Flag antibody. Full-length AdipoR1 (residues 1–375) and the C-terminally truncated mutant (residues 1–370, 1–366 and 1–362) were used in a. AdipoR1 residues 1–375, MYFMAPL (residues 161–167) changed to SGSSGGS (ECL1); residues 1–375, YCS (residues 229–231) changed to GGG (ECL2); residues 1–375, FVKATTV (residues 291–297) changed to SSSGGGS (ECL3); residues 1–375, ECL1 and ECL3 (ECL1/3); and residues 1–375, ECL1, ECL2 and ECL3 (ECL1/2/3) were used in b.

Extended Data Table 1 Data collection and refinement statistics (Molecular replacement)
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Tanabe, H., Fujii, Y., Okada-Iwabu, M. et al. Crystal structures of the human adiponectin receptors. Nature 520, 312–316 (2015). https://doi.org/10.1038/nature14301

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