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Conformational landscape of soluble α-klotho revealed by cryogenic electron microscopyNicholas J. Schnicker 1,2,5? , Zhen Xu 1,5 , Mohammad Amir 3,5 , Lokesh Gakhar 1,4 & Chou-Long Huang 3?α-Klotho (KLA) is a type-1 membranous protein that can associate with fi broblast growth factor receptor (FGFR) to form co-receptor for FGF23. The ectodomain of unassociated KLA is shed as soluble KLA (sKLA) to exert FGFR/FGF23-independent pleiotropic functions. The previously determined X-ray crystal structure of the extracellular region of sKLA in complex with FGF23 and FGFR1c suggests that sKLA functions solely as an on-demand coreceptor for FGF23. To understand the FGFR/FGF23-independent pleiotropic functions of sKLA, we investigated biophysical properties and structure of apo-sKLA. Single particle cryogenic electron microscopy (cryo-EM) revealed a 3.3 Å resolution structure of apo-sKLA that overlays well with its counterpart in the ternary complex with several distinct features. Compared to the ternary complex, the KL2 domain of apo-sKLA is more fl exible. Three-dimensional variability analysis revealed that apo-sKLA adopts conformations with dif f erent KL1-KL2 interdomain bending and rotational angles. Mass photometry revealed that sKLA can form a stable structure with FGFR and/or FGF23 as well as sKLA dimer in solution. Cryo-EM supported the dimeric structure of sKLA. Recent studies revealed that FGF23 contains two KLA-binding sites. Our computational studies revealed that each site binds separate KLA in the dimer. The potential multiple forms and shapes of sKLA support its role as FGFR-independent hormone with pleiotropic functions. The ability of FGF23 to engage two KLA’s simultaneously raises a potential new mechanism of action for FGF23-mediated signaling by the membranous klotho.Keywords Soluble alpha-klotho (sKLA), Fibroblast growth factor (FGF), Cryogenic electron microscopy (cryo-EM), Ternary complex, Monomer, Dimer, 3D variability analysis (3DVA)α-Klotho (KLA) is a type I single-pass transmembrane protein consisting of 1012 amino acids (human Klotho) with a large extracellular region 1 . Th e ectodomain contains two homologous repeats named KL1 and KL2, which are comprised from residues 57–506 and 515–953, respectively, and has multiple N- and O-linked glycosylation sites. Th e ectodomain is followed by the transmembrane-spanning segment and a short 11 amino acids intracellular carboxyl terminus. KLA is abundantly produced in the kidney and several regions in the brain and exerts anti-aging ef f ects 2–4 . Mice homozygous for a hypomorphic klotho allele (kl/kl) die prematurely at around 2–3 months of age. Th e full-length membranous KLA can associate with fi broblast growth factor receptors (FGFR) to form co-receptors for the ligand fi broblast growth factor-23 (FGF23). FGF23 is a bone-derived circulating hormone that is important in calcium and phosphate metabolism 5–7 . KLA-def i cient mice have severe hyperphosphatemia due to defects in the KLA-FGF23-vitamin D regulatory axis 8–10 . Phosphate retention is pivotal for growth retardation and premature death of klotho-def i cient mice; dietary phosphate restriction rescues growth defects and premature death of the mice 8–10 . Of note, the af f i nity between FGF23 and FGFR1c is relatively low (723 nM) 11 . Th e higher af f i nity interactions of KLA with both FGF23 (15 nM) 12 , and FGFR1c (72 nM) 13 facilitates formation of ternary FGF23-FGFR-KLA complexes 14 . Without FGF23, signif i cant fractions of membranous KLA are dissociated from FGFR existing in a free form 14 . Th e ectodomain of free KLA can be cleaved by metalloproteases (ADAM10/17) and released as soluble KLA (sKLA) into the systemic circulation, urine, and cerebrospinal fl uid 15 .1 Protein and Crystallography Facility, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA. 2 Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA. 3 Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA. 4 Department of Biochemistry and Molecular Biology, University of Iowa, Iowa City, IA 52242, USA. 5 Nicholas J. Schnicker, Zhen Xu and Mohammad Amir contributed equally. ? email: nicholas-schnicker@uiowa.edu; chou-long-huang@uiowa.eduOPENScientif i c Reports | (2025) 15:543 1 | https://doi.org/10.1038/s41598-024-84246-xwww.nature.com/scientificreports