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 ‘zip code’-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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Eswarakumar, V. P., Lax, I. & Schlessinger, J. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev. 16, 139–149 (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, 7432–7437 (2007)
Urakawa, I. et al. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature 444, 770–774 (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, 22–29 (2015)
Koshland, D. E. Jr. Stereochemistry and the mechanism of enzymatic reactions. Biol. Rev. Camb. Philos. Soc. 28, 416–436 (1953)
Holm, L. & Rosenström, P. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38, W545–W549 (2010)
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (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, 181–183 (2002)
Yie, J. et al. FGF21 N- and C-termini play different roles in receptor interaction and activation. FEBS Lett. 583, 19–24 (2009)
Micanovic, R. et al. Different roles of N- and C- termini in the functional activity of FGF21. J. Cell. Physiol. 219, 227–234 (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, 605–614 (2016)
Dunshee, D. R. et al. Fibroblast activation protein cleaves and inactivates fibroblast growth factor 21. J. Biol. Chem. 291, 5986–5996 (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, 169–179 (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, 1204–1218 (2007)
Degirolamo, C., Sabbà, C. & Moschetta, A. Therapeutic potential of the endocrine fibroblast growth factors FGF19, FGF21 and FGF23. Nat. Rev. Drug Discov. 15, 51–69 (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, 270–280 (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, 730–743 (2015)
Schlessinger, J. 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)
Pardon, E. et al. A general protocol for the generation of nanobodies for structural biology. Nat. Protoc. 9, 674–693 (2014)
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)
Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
Adams, P. D . et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)
Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51, 2778–2786 (2011)
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.
The authors declare no competing financial interests.
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.
a–c, Size-exclusion chromatography profiles and corresponding Coomassie-stained SDS–PAGE gels of the sKLB−FGFR1cD2D3−FGF21 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 SDS–PAGE 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 KLB–Fc 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 6–8 Å, 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 sKLB–Nb914 complex diffracted to a resolution of 2.2 Å. We also crystallized KLBD1 in complex with Nb914 (c), and collected data to a resolution of 1.7 Å. 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.
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.
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 Kd = 43.5 ± 5.0 nM and Kd = 940 ± 176 nM, respectively. c, d, MST-based competition assay with GST–FGF21CT that contained mutations in regions that interact with site 1 (c) or site 2 (d). Half-maximal inhibitory concentration (IC50) values for wild type, 704 ± 96 nM; D192A, 15,900 ± 6,210 nM; P193A, 7,160 ± 2,350 nM; S204A, 5,990 ± 1,040 nM; S206A, 5,560 ± 1,590 nM; and Y207A, 6,630 ± 1,570 nM. The dots and error bars in panels a–d denote mean and s.d. of ΔFnorm (n = 3 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.
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.
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.
Figures that contain the data are indicated.
About this article
Cite this article
Lee, S., Choi, J., Mohanty, J. et al. Structures of β-klotho reveal a ‘zip code’-like mechanism for endocrine FGF signalling. Nature 553, 501–505 (2018). https://doi.org/10.1038/nature25010
Optimization of Peptide Inhibitors of β-Klotho as Antagonists of Fibroblast Growth Factors 19 and 21
ACS Pharmacology & Translational Science (2020)
Journal of Hepatology (2020)
The FGF-21 genetic variants rs838133 and rs838145 are associated with high salt intake in the Emirati population
Journal of Advanced Research (2020)
Scientific Reports (2020)
Novel Abs targeting the N‐terminus of fibroblast growth factor 19 inhibit hepatocellular carcinoma growth without bile‐acid‐related side‐effects
Cancer Science (2020)