Article | Published:

α-Klotho is a non-enzymatic molecular scaffold for FGF23 hormone signalling

Nature volume 553, pages 461466 (25 January 2018) | Download Citation

Abstract

The ageing suppressor α-klotho binds to the fibroblast growth factor receptor (FGFR). This commits FGFR to respond to FGF23, a key hormone in the regulation of mineral ion and vitamin D homeostasis. The role and mechanism of this co-receptor are unknown. Here we present the atomic structure of a 1:1:1 ternary complex that consists of the shed extracellular domain of α-klotho, the FGFR1c ligand-binding domain, and FGF23. In this complex, α-klotho simultaneously tethers FGFR1c by its D3 domain and FGF23 by its C-terminal tail, thus implementing FGF23–FGFR1c proximity and conferring stability. Dimerization of the stabilized ternary complexes and receptor activation remain dependent on the binding of heparan sulfate, a mandatory cofactor of paracrine FGF signalling. The structure of α-klotho is incompatible with its purported glycosidase activity. Thus, shed α-klotho functions as an on-demand non-enzymatic scaffold protein that promotes FGF23 signalling.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from $8.99

All prices are NET prices.

Accessions

Primary accessions

Protein Data Bank

References

  1. 1.

    et al. Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism. J. Clin. Invest. 113, 561–568 (2004)

  2. 2.

    et al. FGF23 decreases renal NaPi-2a and NaPi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor 1. Am. J. Physiol. Renal Physiol. 297, F282–F291 (2009)

  3. 3.

    & Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010)

  4. 4.

    et al. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell 6, 743–750 (2000)

  5. 5.

    , & Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev. 16, 107–137 (2005)

  6. 6.

    & Exploring mechanisms of FGF signalling through the lens of structural biology. Nat. Rev. Mol. Cell Biol. 14, 166–180 (2013)

  7. 7.

    et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 390, 45–51 (1997)

  8. 8.

    & Structural and sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7, 637–644 (1997)

  9. 9.

    et al. Klotho coreceptors inhibit signaling by paracrine fibroblast growth factor 8 subfamily ligands. Mol. Cell. Biol. 32, 1944–1954 (2012)

  10. 10.

    et al. Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23–FGFR–Klotho complex formation. Proc. Natl Acad. Sci. USA 107, 407–412 (2010)

  11. 11.

    et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 444, 770–774 (2006)

  12. 12.

    et al. Regulation of fibroblast growth factor-23 signaling by klotho. J. Biol. Chem. 281, 6120–6123 (2006)

  13. 13.

    et al. Immunohistochemical localization of Klotho protein in brain, kidney, and reproductive organs of mice. Cell Struct. Funct. 29, 91–99 (2004)

  14. 14.

    et al. Shedding of klotho by ADAMs in the kidney. Am. J. Physiol. Renal Physiol. 309, F359–F368 (2015)

  15. 15.

    et al. The kidney is the principal organ mediating klotho effects. J. Am. Soc. Nephrol. 25, 2169–2175 (2014)

  16. 16.

    , , , & Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17. Proc. Natl Acad. Sci. USA 104, 19796–19801 (2007)

  17. 17.

    et al. Secreted Klotho protein in sera and CSF: implication for post-translational cleavage in release of Klotho protein from cell membrane. FEBS Lett. 565, 143–147 (2004)

  18. 18.

    et al. Identification of the human klotho gene and its two transcripts encoding membrane and secreted klotho protein. Biochem. Biophys. Res. Commun. 242, 626–630 (1998)

  19. 19.

    et al. Structure of the mouse klotho gene and its two transcripts encoding membrane and secreted protein. FEBS Lett. 424, 6–10 (1998)

  20. 20.

    et al. Suppression of aging in mice by the hormone Klotho. Science 309, 1829–1833 (2005)

  21. 21.

    , , & Fibroblast growth factor 23 and Klotho: physiology and pathophysiology of an endocrine network of mineral metabolism. Annu. Rev. Physiol. 75, 503–533 (2013)

  22. 22.

    et al. The β-glucuronidase klotho hydrolyzes and activates the TRPV5 channel. Science 310, 490–493 (2005)

  23. 23.

    et al. Removal of sialic acid involving Klotho causes cell-surface retention of TRPV5 channel via binding to galectin-1. Proc. Natl Acad. Sci. USA 105, 9805–9810 (2008)

  24. 24.

    et al. Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. FASEB J. 24, 3438–3450 (2010)

  25. 25.

    et al. α-Klotho as a regulator of calcium homeostasis. Science 316, 1615–1618 (2007)

  26. 26.

    et al. Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase. J. Biol. Chem. 282, 30889–30900 (2007)

  27. 27.

    et al. Klotho lacks an FGF23-independent role in mineral homeostasis. J. Bone Miner. Res. 32, 2049–2061 (2017)

  28. 28.

    et al. C-terminal tail of FGF19 determines its specificity toward Klotho co-receptors. J. Biol. Chem. 283, 33304–33309 (2008)

  29. 29.

    et al. Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells. Mol. Cell. Biol. 12, 240–247 (1992)

  30. 30.

    , , , & Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature 407, 1029–1034 (2000)

  31. 31.

    et al. Towards a resolution of the stoichiometry of the fibroblast growth factor (FGF)–FGF receptor–heparin complex. J. Mol. Biol. 339, 821–834 (2004)

  32. 32.

    et al. βKlotho is required for metabolic activity of fibroblast growth factor 21. Proc. Natl Acad. Sci. USA 104, 7432–7437 (2007)

  33. 33.

    et al. Tissue-specific expression of βKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21. J. Biol. Chem. 282, 26687–26695 (2007)

  34. 34.

    et al. Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis. Genes Dev. 17, 1581–1591 (2003)

  35. 35.

    et al. FGF21 induces PGC-1alpha and regulates carbohydrate and fatty acid metabolism during the adaptive starvation response. Proc. Natl Acad. Sci. USA 106, 10853–10858 (2009)

  36. 36.

    et al. Digenic mutations account for variable phenotypes in idiopathic hypogonadotropic hypogonadism. J. Clin. Invest. 117, 457–463 (2007)

  37. 37.

    et al. Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat. Genet. 26, 345–348 (2000)

  38. 38.

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

  39. 39.

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

  40. 40.

    et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

  41. 41.

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

  42. 42.

    et al. Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members. Mol. Cell. Biol. 27, 3417–3428 (2007)

  43. 43.

    , , & Structural basis for FGF receptor dimerization and activation. Cell 98, 641–650 (1999)

  44. 44.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  45. 45.

    et al. βKlotho is required for fibroblast growth factor (FGF) 21 signaling through FGF receptor (FGFR) 1c and FGFR3c. Mol. Endocrinol. 22, 1006–1014 (2008)

  46. 46.

    et al. FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides. Science 268, 432–436 (1995)

  47. 47.

    . et al. Regulation of receptor binding specificity of FGF9 by an autoinhibitory homodimerization. Structure 25, 1325–1336 (2017)

  48. 48.

    & Molecular mechanisms of fibroblast growth factor signaling in physiology and pathology. Cold Spring Harb. Perspect. Biol. 5, a015958 (2013)

  49. 49.

    , , , & Plasticity in interactions of fibroblast growth factor 1 (FGF1) N terminus with FGF receptors underlies promiscuity of FGF1. J. Biol. Chem. 287, 3067–3078 (2012)

  50. 50.

    et al. Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain. Genes Dev 20, 185–198 (2006)

  51. 51.

    , , , & Cooperative dimerization of fibroblast growth factor 1 (FGF1) upon a single heparin saccharide may drive the formation of 2:2:1 FGF1.FGFR2c.heparin ternary complexes. J. Biol. Chem. 280, 42274–42282 (2005)

  52. 52.

    et al. Evidence that heparin saccharides promote FGF2 mitogenesis through two distinct mechanisms. J. Biol. Chem. 283, 13001–13008 (2008)

  53. 53.

    et al. Fibroblast activation protein cleaves and inactivates fibroblast growth factor 21. J. Biol. Chem. 291, 5986–5996 (2016)

Download references

Acknowledgements

We thank N. J. Cowan for critically reading and editing the manuscript, and C.-S. Huang for help with diffraction data processing with XDS. This work was primarily supported by NIH grant R01 DE13686 (to M.M.). Support was also provided by National Key R&D Program of China (#2017YFA0506000 to X.L.). Funding for mouse studies was provided by R01 DK092461, P30 DK079328 (to O.W.M.), and R01 DK091392 (to M.C.H). Beamlines at the Northeastern Collaborative Access Team (NE-CAT) facility at the Advanced Photon Source of Argonne National Laboratory are primarily funded by NIH NIGMS and member institutions.

Author information

Author notes

    • Gaozhi Chen
    •  & Yang Liu

    These authors contributed equally to this work.

Affiliations

  1. Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China

    • Gaozhi Chen
    • , Lili Fu
    • , Guang Liang
    •  & Xiaokun Li
  2. Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, New York 10016, USA

    • Gaozhi Chen
    • , Yang Liu
    • , Regina Goetz
    • , Lili Fu
    •  & Moosa Mohammadi
  3. New York Structural Biology Center, New York, New York 10027, USA

    • Seetharaman Jayaraman
  4. Departments of Internal Medicine and Physiology, and Charles and Jane Pak Center of Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Ming-Chang Hu
    •  & Orson W. Moe

Authors

  1. Search for Gaozhi Chen in:

  2. Search for Yang Liu in:

  3. Search for Regina Goetz in:

  4. Search for Lili Fu in:

  5. Search for Seetharaman Jayaraman in:

  6. Search for Ming-Chang Hu in:

  7. Search for Orson W. Moe in:

  8. Search for Guang Liang in:

  9. Search for Xiaokun Li in:

  10. Search for Moosa Mohammadi in:

Contributions

G.C. purified and crystallized the ternary complex, analysed the crystal structure, generated SEC–MALS data (Figs 4a, 5a, b, f), cell-based data (Fig. 4), enzyme and thermostability assay data (Fig. 2c), and participated in the design of experiments and the writing/revising of the manuscript. Y.L. helped with data collection and analysis of the crystal structure, generated cell-based data (Fig. 5), and participated in manuscript revision. R.G. established expression and purification protocols for the ternary complex, performed ternary complex characterization, analysed mouse data, and participated in editing and revising the manuscript. L.F. generated expression constructs for FGF23, FGFR1cecto, α-klothoecto and their structure-based mutated forms, and helped with ternary complex purification. S.J. assisted with diffraction data collection and performed excitation/emission scanning of the FGF23–FGFR1cecto–α-klothoecto crystal (Extended Data Fig. 2c). M.-C.H. and O.W.M. generated the mouse data (Extended Data Figs 1c, d and 7a, b). G.L. and X.L. (mentors of G.C. and L.F.) participated in manuscript revision. M.M. developed and directed the project, solved, refined, analysed and interpreted the crystal structure of the ternary complex, and wrote the manuscript.

Competing interests

O.W.M. has done paid consultation for AbbVie, Allena, Amgen, and Tricida. He also sits on the board of Klotho Therapeutics. All of the other authors have no competing financial interests to declare.

Corresponding authors

Correspondence to Xiaokun Li or Moosa Mohammadi.

Reviewer Information Nature thanks M. Kuro-o 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.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figure 1 and Supplementary Tables 1-2.

  2. 2.

    Life Sciences Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature25451

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.