A systematic dissection of sequence elements determining β-Klotho and FGF interaction and signaling

Endocrine fibroblast growth factors (FGFs) require Klotho transmembrane proteins as necessary co-receptors to activate FGF receptor (FGFR) signaling. In particular, FGF19 and FGF21 function through β-Klotho to regulate glucose and lipid metabolism. Recent research has focused on elucidating how these two FGFs interact with β-Klotho and FGFRs to activate downstream signaling. In this study, using hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS), we identified regions on the β-Klotho protein that likely participate in ligand interaction, and vice versa. Alanine and arginine mutagenesis were carried out to further probe the contributions of individual residues to receptor/ligand interactions. Using biochemical and cell-based signaling assays with full-length proteins, we show that both the KL1 and KL2 domains of β-Klotho participate in ligand interaction, and these binding sites on β-Klotho are shared by FGF19 and FGF21. In addition, we show that two highly conserved regions in the C-terminal tail of FGF19 and FGF21 are responsible for interaction with the co-receptor. Our results are consistent with recent publications on the crystal structures of the Klotho proteins and provide insight into how endocrine FGFs interact with co-receptors for signal transduction.