αKlotho Forms Dimer for Fibroblast Growth Factor 23 (FGF-23) Binding via Two Tandem Repeat Binding Sites: Novel Signalling Mechanism for FGF-23-Fibroblast Growth Factor Receptor (FGFR)-Klotho Complex

Background: Fibroblast growth factor-23 (FGF23) contains 24 amino acids (a.a) signal peptide and a.a. 25-251 circulating peptide that binds α-Klotho (KLA) and FGFR coreceptor to regulate 1,25-VitD3 synthesis and phosphate homeostasis. Early studies showed that a.a. 25-205 of FGF23 lacking the last 46 a.a can bind the co-receptor and has in vivo activities. The KLA-binding site resides in a.a. 180-205, has a critical DPL (Asp-Pro-Leu) motif. Recently, the last 46 a.a of FGF23 was reported to contain a second KLA-binding site (a.a. 212-239; “R2”) homologous to the first binding site (a.a. 180-205; “R1”) and has same conserved “DPL” motif. R1 and R2 bind KLA with equal affinity. The physiological role of the tandem binding sites on FGF23 yet is unknown. Methods: Cryo-EM and molecular modelling were performed. Knockin (KI) mice carrying FGF23 mutations were studied. Results: Cryo-EM structural analysis revealed apo-KLA adopted dimers in solution. Modelling revealed that KLA dimer binds one FGF23 with R1 and R2 interacting with KLA-1 and KLA-2, respectively. With FGFR, the stoichiometry of ternary complex is FGF23-FGFR-(KLA)2 (1:1:2). Interestingly, R1 and R2 bind to different surface of KLA-1 and -2. This arrangement is feasible as the frontal plane of the two KLA’s in the dimer adopt different tilt angles perpendicular to the cell membrane. The critical DPL motif in both R1 and R2 closely match the conformation of the previously solved FGF23-FGFR-KLA complex. To test in vivo relevance of KLA dimers, we generated mice carrying mutation that disrupt R1 binding to KLA (D188A). Mice homozygous for D188A-knockin were born alive and grew normally as wildtype (WT) mice. Serum phosphate and VitD3 levels were not significant different between KI and WT in normal diet. Experiments are ongoing to analyse serum chemistry under high and low phosphate diets. Additional KI mice carrying R2 mutation, double R1/R2 mutation, and mutation disrupting KLA dimer formation are ongoing. Conclusion: FGF23 is a bivalent ligand with the R2 repeat playing a crucial role in the formation of FGF23-FGFR1-(KLA)2 complexes. With the recent report that FGFR forms asymmetrical dimers, we propose a new model of FGF23 signaling through formation of FGF23-(FGFR)2-(KLA)2 complex (i.e., 1:2:2 stoichiometry). This complex confers enhanced FGF23 signaling. Funding: NIDDK Support