Abstract
Canonical fibroblast growth factors (FGFs) activate FGF receptors (FGFRs) through paracrine or autocrine mechanisms in a process that requires cooperation with heparan sulfate proteoglycans, which function as co-receptors for FGFR activation1,2. By contrast, endocrine FGFs (FGF19, FGF21 and FGF23) are circulating hormones that regulate critical metabolic processes in a variety of tissues3,4. FGF19 regulates bile acid synthesis and lipogenesis, whereas FGF21 stimulates insulin sensitivity, energy expenditure and weight loss5. Endocrine FGFs signal through FGFRs in a manner that requires klothos, which are cell-surface proteins that possess tandem glycosidase domains3,4. Here we describe the crystal structures of free and ligand-bound β-klotho extracellular regions that reveal the molecular mechanism that underlies the specificity of FGF21 towards β-klotho and demonstrate how the FGFR is activated in a klotho-dependent manner. β-Klotho serves as a primary âzip codeâ-like receptor that acts as a targeting signal for FGF21, and FGFR functions as a catalytic subunit that mediates intracellular signalling. Our structures also show how the sugar-cutting enzyme glycosidase has evolved to become a specific receptor for hormones that regulate metabolic processes, including the lowering of blood sugar levels. Finally, we describe an agonistic variant of FGF21 with enhanced biological activity and present structural insights into the potential development of therapeutic agents for diseases linked to endocrine FGFs.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 /Â 30Â days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Eswarakumar, V. P., Lax, I. & Schlessinger, J. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev. 16, 139â149 (2005)
Belov, A. A. & Mohammadi, M. Molecular mechanisms of fibroblast growth factor signaling in physiology and pathology. Cold Spring Harb. Perspect. Biol. 5, a015958 (2013)
Ogawa, Y. et al. βKlotho is required for metabolic activity of fibroblast growth factor 21. Proc. Natl Acad. Sci. USA 104, 7432â7437 (2007)
Urakawa, I. et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 444, 770â774 (2006)
Owen, B. M., Mangelsdorf, D. J. & Kliewer, S. A. Tissue-specific actions of the metabolic hormones FGF15/19 and FGF21. Trends Endocrinol. Metab. 26, 22â29 (2015)
Koshland, D. E. Jr. Stereochemistry and the mechanism of enzymatic reactions. Biol. Rev. Camb. Philos. Soc. 28, 416â436 (1953)
Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545âW549 (2010)
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774â797 (2007)
Hayward, S. & Lee, R. A. Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50. J. Mol. Graph. Model. 21, 181â183 (2002)
Yie, J. et al. FGF21 N- and C-termini play different roles in receptor interaction and activation. FEBS Lett. 583, 19â24 (2009)
Micanovic, R. et al. Different roles of N- and C- termini in the functional activity of FGF21. J. Cell. Physiol. 219, 227â234 (2009)
Hecht, R. et al. Rationale-based engineering of a potent long-acting FGF21 analog for the treatment of type 2 diabetes. PLoS ONE 7, e49345 (2012)
Zhen, E. Y., Jin, Z., Ackermann, B. L., Thomas, M. K. & Gutierrez, J. A. Circulating FGF21 proteolytic processing mediated by fibroblast activation protein. Biochem. J. 473, 605â614 (2016)
Dunshee, D. R. et al. Fibroblast activation protein cleaves and inactivates fibroblast growth factor 21. J. Biol. Chem. 291, 5986â5996 (2016)
Coppage, A. L. et al. Human FGF-21 is a substrate of fibroblast activation protein. PLoS ONE 11, e0151269 (2016)
Chuenchor, W. et al. The structural basis of oligosaccharide binding by rice BGlu1 beta-glucosidase. J. Struct. Biol. 173, 169â179 (2011)
Isorna, P. et al. Crystal structures of Paenibacillus polymyxa β-glucosidase B complexes reveal the molecular basis of substrate specificity and give new insights into the catalytic machinery of family I glycosidases. J. Mol. Biol. 371, 1204â1218 (2007)
Degirolamo, C., Sabbà , C. & Moschetta, A. Therapeutic potential of the endocrine fibroblast growth factors FGF19, FGF21 and FGF23. Nat. Rev. Drug Discov. 15, 51â69 (2016)
Kharitonenkov, A. et al. Rational design of a fibroblast growth factor 21-based clinical candidate, LY2405319. PLoS ONE 8, e58575 (2013)
Huang, Z. et al. A better anti-diabetic recombinant human fibroblast growth factor 21 (rhFGF21) modified with polyethylene glycol. PLoS ONE 6, e20669 (2011)
Huang, J. et al. Development of a novel long-acting antidiabetic FGF21 mimetic by targeted conjugation to a scaffold antibody. J. Pharmacol. Exp. Ther. 346, 270â280 (2013)
Foltz, I. N. et al. Treating diabetes and obesity with an FGF21-mimetic antibody activating the βKlotho/FGFR1c receptor complex. Sci. Transl. Med. 4, 162ra153 (2012)
Luo, J. et al. A nontumorigenic variant of FGF19 treats cholestatic liver diseases. Sci. Transl. Med. 6, 247ra100 (2014)
Kolumam, G. et al. Sustained brown fat stimulation and insulin sensitization by a humanized bispecific antibody agonist for fibroblast growth factor receptor 1/βklotho complex. EBioMedicine 2, 730â743 (2015)
Schlessinger, J. et al. Crystal structure of a ternary FGFâFGFRâheparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell 6, 743â750 (2000)
Pardon, E. et al. A general protocol for the generation of nanobodies for structural biology. Nat. Protoc. 9, 674â693 (2014)
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307â326 (1997)
Kabsch, W. Xds. Acta Crystallogr. D 66, 125â132 (2010)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658â674 (2007)
Adams, P. D . et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213â221 (2010)
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486â501 (2010)
Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligandâprotein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778â2786 (2011)
Acknowledgements
The NSLS-SSRL is supported by P41GM111244, P41GM103393, DE-SC0012704 and by DE-AC02-76SF00515. We thank NE-CAT (P41 GM103403) and APS (DE-AC02-06CH11357). This research was also supported by NIH grant 1S10OD018007 and NIH Award S10RR026992-0110. J.St. thanks INSTRUCT (ESFRI, FWO) for financial support and I. Aboutaleb for technical assistance.
Author information
Authors and Affiliations
Contributions
S.L. designed, performed experiments and determined the crystal structures. J.C., J.M. and F.T. provided technical support. E.P. and J.St. generated nanobodies. L.P.S. and I.L. designed and analysed cell-based experiments. S.L., M.A.L. and J.Sc. designed experiments, analysed data and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Additional information
Reviewer Information Nature thanks N. Jura, K. White, H. E. Xu 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 figures and tables
Extended Data Figure 1 Expression, purification and crystallization of β-klotho extracellular domain.
aâc, Size-exclusion chromatography profiles and corresponding Coomassie-stained SDSâPAGE gels of the sKLBâFGFR1cD2D3âFGF21 ternary complex (green) or sKLB alone (blue) (a), sKLB in complex with Nb914 (b) and KLBD1 in complex with Nb914 (c). The chromatograms and the SDSâPAGE gels shown are representatives of at least three independent preparations with similar results. A secreted protein composed of the extracellular domain of KLB fused to the Fc region of human IgG1 was produced by HEK293 EBNA cells. Following purification using a protein A agarose resin, the KLBâFc fusion protein was subjected to proteolytic cleavage. sKLB was further purified using ion exchange and size-exclusion chromatography. Multiple crystallization trials with the ternary complex formed by sKLB, FGF21 and FGFR1cD2D3 (a, green) failed to yield diffraction-quality crystals. However, a preparation of sKLB bound to a nanobody Nb914 (b) yielded crystals that diffracted X-rays to a resolution of 6â8âà , and these were further improved by mutating two of the eleven potential N-glycosylation sites in sKLB (Asn308 and Asn611) to glutamine residues. The resulting crystals of an sKLBâNb914 complex diffracted to a resolution of 2.2âà . We also crystallized KLBD1 in complex with Nb914 (c), and collected data to a resolution of 1.7âà . The structure of KLBD1 was first solved by molecular replacement using the coordinates of a structure of human cytosolic β-glucosidase (PDB code: 2ZOX) and the coordinates of a nanobody structure (PDB code: 5IMK, chain B) as search models. The structure of sKLB was subsequently determined by molecular replacement using the KLBD1 coordinates as a search model.
Extended Data Figure 2 Domain diagram of sKLB structure and the location of cysteine residues.
a, Secondary structure elements (H for helix (green) and S for sheet (red)) are designated by numbers on the basis of the principal elements for the (β/α)8 fold. Dashed lines depict disordered loops that are not modelled in the structure. b, Seven of the ten cysteine residues in the extracellular region were successfully modelled in the sKLB structure. With the exception of the disulfide bond between Cys576 and Cys625, the structure shows that these cysteine residues are reduced and do not form disulfide bridges. Moreover, determination of the distances between each pair of cysteines indicates that most are too far apart to form intramolecular disulfide bonds. However, we cannot rule out the possibility that Cys976 located in the C-terminal region of sKLB, which could not be modelled owing to weak electron density, may form a disulfide bond with the nearby Cys523. There is no evidence for the formation of intermolecular disulfide bonds between β-klotho and the closely associated FGFR, FGF19 or FGF21 proteins, whose cysteines all form well-characterized intramolecular disulfide bonds. The functional consequences of the presence of reduced cysteines in β-klotho are currently unknown.
Extended Data Figure 3 Unique structural features of sKLB.
a, Interaction of H6a (green) with the pseudo-substrate binding pocket in D1 of sKLB. Glu416, the pseudo-catalytic glutamic acid residue in D1, is located on the bottom of the pocket and is also highlighted. b, Interaction of H0 (green) with the nearby structural elements in D1 of sKLB. c, Interface between D1 (blue) and D2 (green) of sKLB, highlighting amino acids and structural elements as well as polar interactions (red dotted lines) between the domains.
Extended Data Figure 4 Details of interactions between sKLB and FGF21CT, and conformational changes upon ligand binding.
a, Interactions between amino acid residues in sKLB (green) and FGF21CT (salmon) in the areas of sites 1 and 2 are indicated. b, Diagram of amino-acid-specific interactions between sKLB and FGF21CT within sites 1 and 2. The figure was generated using Ligplot+32. c, Structure of sKLB (green) in complex with FGF21CT (salmon) shown as a surface representation. d, Structure of ligand-free sKLB (blue) is overlaid onto the structure of sKLB (green) bound to FGF21CT (salmon, ball-and-stick).
Extended Data Figure 5 Amino acid sequence alignments of C-terminal regions of human FGF19 and FGF21.
Residues Asp-Pro, which are critical in maintaining multi-turn elements, are highlighted in blue, and the sugar-mimicking motif Ser-Pro-Ser is highlighted in yellow. The sequence alignment reveals close sequence similarity between the C-terminal tails of FGF21 and FGF19 that is consistent with the similar binding characteristics of FGF21 and FGF19 and their isolated C-terminal regions to β-klotho. The sugar-mimicking motif in FGF21, Ser205-Pro206-Ser207, is conserved in FGF19 (Ser211-Pro212-Ser213). The sequence Asp192-Pro193, in the region of FGF21CT that binds to site 1 of β-klotho by stabilizing intramolecular hydrogen bonds that maintain a turn in the bound configuration of FGF21CT, is also highlighted. This sequence is conserved in FGF19 (Asp198-Pro199), which suggests that intramolecular interactions similar to those responsible for mediating consecutive turns in FGF19CT may also bind to site-1 of β-klotho. Because many of the intramolecular interactions within FGF21CT bound to β-klotho take place between main-chain atoms (as observed in typical β-turn structures), the presence of only a few key amino acid sequences such as Asp198-Pro199 may be sufficient to generate multi-turn elements in FGF19CT that are similar to those observed in the crystal structure of FGF21CT bound to β-klotho.
Extended Data Figure 6 Validation of FGF21-binding interface to β-klotho by ligand-binding and cell-stimulation experiments.
a, b, MST-based binding affinity measurements of (a) FGF21 to sKLB (a) and FGFR1cD2D3 to sKLB (b) that yielded Kdâ=â43.5â±â5.0ânM and Kdâ=â940â±â176ânM, respectively. c, d, MST-based competition assay with GSTâFGF21CT that contained mutations in regions that interact with site 1 (c) or site 2 (d). Half-maximal inhibitory concentration (IC50) values for wild type, 704â±â96ânM; D192A, 15,900â±â6,210ânM; P193A, 7,160â±â2,350ânM; S204A, 5,990â±â1,040ânM; S206A, 5,560â±â1,590ânM; and Y207A, 6,630â±â1,570ânM. The dots and error bars in panels aâd denote mean and s.d. of ÎFnorm (nâ=â3 independent samples). Individual experimental data are plotted in Extended Data Fig. 9. e, Location of mutated amino acid residues (yellow) in sKLB (green) occupied by FGF21 (salmon) that were analysed in f and g. f, g, Stably transfected L6 cells co-expressing FGFR1c together with wild-type or β-klotho mutants were stimulated with either FGF21 or FGF1 (control) and analysed for FGFR1c activation by monitoring tyrosine phosphorylation of FGFR1c. Lysates of ligand-stimulated or unstimulated cells were subjected to immunoprecipitation with anti-FGFR1 antibodies, followed by immunoblotting with either anti-pTyr or anti-FGFR1 antibodies.
Extended Data Figure 7 β-Klotho is required for FGFR1c-mediated signalling induced by FGF21.
a, b, L6 cells that expressed either FGFR1c alone (a) or FGFR1c together with β-klotho (b) were stimulated with a range of concentrations of FGF1 or FGF21, and phosphotyrosine (pTyr) levels of FGFR were monitored by immunoprecipitation with anti-FGFR1 antibodies, followed by immunoblotting with anti-pTyr antibodies.
Extended Data Figure 8 MAP kinase stimulation induced by wild-type or mutant FGF21.
L6 cells that co-expressed β-klotho and FGFR1c were stimulated with wild-type FGF21 (top) or FGF21(R203W/L194F) (bottom), and phosphorylation levels of MAP kinase in cell lysates were monitored.
Extended Data Figure 9 MST data with individual data points.
Figures that contain the data are indicated.
Supplementary information
Supplementary Information
This file contains a Supplementary Discussion, Supplementary References and Supplementary Figure 1. (PDF 2250 kb)
Rights and permissions
About this article
Cite this article
Lee, S., Choi, J., Mohanty, J. et al. Structures of β-klotho reveal a âzip codeâ-like mechanism for endocrine FGF signalling. Nature 553, 501â505 (2018). https://doi.org/10.1038/nature25010
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature25010
This article is cited by
-
Mitochondrial stress: a key role of neuroinflammation in stroke
Journal of Neuroinflammation (2024)
-
A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel hostâpathogen interactions
Malaria Journal (2024)
-
FGF19 increases mitochondrial biogenesis and fusion in chondrocytes via the AMPKα-p38/MAPK pathway
Cell Communication and Signaling (2023)
-
Heating-mediated purification of active FGF21 and structure-based design of its variant with enhanced potency
Scientific Reports (2023)
-
Regulation of FGF23 production and phosphate metabolism by boneâkidney interactions
Nature Reviews Nephrology (2023)
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.