Extended Data Fig. 4: FGF23 N-terminus experiences a major conformational change in the quaternary complex.

A, Left and Center: Cartoon representations of the FGF23-FGFR^S and FGFR^P-αKlotho components of the quaternary complex shown in the orientation obtained via alignment of D2 domains of FGFR^S and FGFR^P. Right: Superimposition of FGF23-FGFR^S and FGFR^P-αKlotho components via aligning receptors’ D3 domains. Note that FGF23 N-terminus and αKlotho RBA engage the equivalent hydrophobic grooves in D3 of FGFR^S and FGFR^P chains. b–d, FGF23 N-terminus undergoes a major conformational change in FGF23–FGFR–αKlotho–HS quaternary complexes. Cartoon and surface (only for FGF23 component) representation of HS-free ternary complex (that is, X-ray structure) (b) and HS-induced quaternary complex (that is, cryo-EM structures) (c) in the same orientation obtained via alignment of their FGF23 chains. (d) Cartoon representation of an overlay between FGF23-FGFR1c from FGF23–FGFR1c–αKlotho ternary complex and FGF23-FGFR1c^P from quaternary complex via FGF23 alignment. Note the dramatic difference in the positions of FGF23 N-terminus between the two structures. e, Electron densities of FGF23 N-terminus in cryo-EM structures of FGF23–FGFR1c–αKlotho–HS quaternary complexes at the indicated contour levels. Note that the electron densities for FGF23 N-terminus are weak and patchy. f–i, MD simulation data show that FGF23 N-terminus engages D3 of FGFR1c^S. f, Left: Cartoon representation of the cryo-EM structure of FGF23-FGFR1c-αKlotho-HS quaternary complex. FGF23, FGFR1c^P, FGFR1c^S and αKlotho are in orange, green, cyan, and blue, respectively. Right: Zoomed-in view of the boxed region (left) showing conformational progression of the N-terminal tail of FGF23 (that is, Y25 to W36) and D3 of FGFR1c^S in a 300 ns MD simulation trajectory denoted by a color transition from the orange (0 ns) to blue (300 ns) in 60 ns intervals. FGF23 N-terminus interacts with the three stranded βC: βF: βG sheet in FGFR1c^S D3 domain. g-h, Changes in RMSD (g) and Root Mean Square Fluctuation (RMSF, h), respectively, of N-terminal tail of FGF23 during MD simulation. Note that RMSD of N-terminal residues of FGF23 stabilized around 4 Å after 120 ns. Importantly, residues at the distal and proximal ends of FGF23 N-terminus exhibited largest and smallest RMSF, respectively, mirroring their respective cryo-EM electron densities (compare e and h). i, Changes in the distances of four selected contact pairs (Y25–L342^S, S29–R254^S, L31–A259^S and L32–I256^S) between N-terminal residues of FGF23 and residues in D3 of FGFR1c^S. The distances of Y25–L342^S, L31–A259^S, and L32–I256^S hydrophobic residue pairs fluctuated around 5 Å indicative of formation of hydrophobic contacts between these residue pairs. Likewise, the pairwise distance for S29-R254^S fluctuated around 4 Å after 120 ns indicative of hydrogen bonding between side chains of this residue pair.