Extended Data Figure 5: α-Klotho is the first non-enzymatic scaffold among TIM barrel proteins.

a, Structure-based sequence alignment of TIM barrels of α-klotho KL1 and KL2 domains and KLrP. Most glycoside hydrolases (GH), a functionally diverse group of enzymes that cleave glycosidic bonds of complex carbohydrates on glycoproteins⁸, adopt a TIM barrel fold. Locations and lengths of TIM barrel β-strands and α-helices are indicated above the alignment. Among GH family 1 members of the klotho subfamily, only KLrP has a verified glycosylceramidase activity²⁶, and Glu165 and Glu373 are its catalytically essential glutamic acids. KLrP residues coloured cyan participate in substrate recognition/hydrolysis. α-Klotho residues coloured red bind FGF23, and α-klotho residues of the KL2 β1α1 loop (purple box) coloured purple interact with the FGFR1c D3 domain. b, Superimposition of KL1 C_α trace (grey/cyan) onto that of KLrP (grey/yellow). Superimposition root mean square deviation (r.m.s.d.) value is 1.08 Å. Structurally most divergent regions between KL1 and KLrP are in cartoon representation. Glucose moiety and aliphatic chains of glucosylceramide (KLrP substrate) are in sticks with carbon in black (glucose) or green/cyan/pink (aliphatic chains). Catalytically essential Glu165 in KLrP is replaced by an asparagine in KL1. Hydrophobic residues from KL1 β6α6 loop occupy the pocket that accommodates the aliphatic chains of glucosylceramide in KLrP. The KL1 N terminus supports KL1–KL2 cleft formation (Extended Data Fig. 2b) and KL1 β6α6 loop conformation contributes to a key portion of the binding pocket in this cleft for the FGF23 C-terminal tail (Fig. 3c). c, d, Superimposition of KL2 C_α trace (grey/blue) onto that of KLrP (grey/yellow). Superimposition r.m.s.d. value is 1.37 Å. Structurally divergent β1α1 (c), β6α6 and β8α8 (d) loops of KL2 and KLrP are rendered in cartoon. β1α1 loop in KL2 is disengaged from the central TIM barrel and stretches away from it by as much as 35 Å. Catalytically essential Glu373 in KLrP is replaced by a serine in KL2. KLrP residues from β6α6 and β8α8 loops bind glucosylceramide (KLrP substrate); for example, Trp345 in the β6α6 loop and Glu424 and Trp425 in the β8α8 loop. Sequence divergence (a) and altered loop conformations are incompatible with glucosylceramide coordination by KL2. β1α1, β6α6 and β8α8 loops lie at the rim of the catalytic mouth in the TIM barrel (see Fig. 2b). Divergent conformations of these three loops in KL2 result in notable widening of the central barrel cavity in KL2, which merges with the KL1–KL2 cleft to form an expansive basin that accommodates the distal portion of the FGF23 C-terminal tail.