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- PDB-8uf8: Cryo-EM structure of alpha-Klotho -

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Basic information

Entry
Database: PDB / ID: 8uf8
TitleCryo-EM structure of alpha-Klotho
ComponentsKlotho
KeywordsSIGNALING PROTEIN / protein binding
Function / homology
Function and homology information


norepinephrine biosynthetic process / beta-glucuronidase / FGFR1c and Klotho ligand binding and activation / positive regulation of MAPKKK cascade by fibroblast growth factor receptor signaling pathway / beta-glucuronidase activity / fibroblast growth factor receptor binding / vitamin D binding / Downstream signaling of activated FGFR1 / Phospholipase C-mediated cascade: FGFR1 / energy reserve metabolic process ...norepinephrine biosynthetic process / beta-glucuronidase / FGFR1c and Klotho ligand binding and activation / positive regulation of MAPKKK cascade by fibroblast growth factor receptor signaling pathway / beta-glucuronidase activity / fibroblast growth factor receptor binding / vitamin D binding / Downstream signaling of activated FGFR1 / Phospholipase C-mediated cascade: FGFR1 / energy reserve metabolic process / response to vitamin D / negative regulation of systemic arterial blood pressure / response to angiotensin / beta-glucosidase activity / PI-3K cascade:FGFR1 / fibroblast growth factor binding / PI3K Cascade / fibroblast growth factor receptor signaling pathway / positive regulation of bone mineralization / SHC-mediated cascade:FGFR1 / calcium ion homeostasis / FRS-mediated FGFR1 signaling / response to activity / determination of adult lifespan / Negative regulation of FGFR1 signaling / hormone activity / Constitutive Signaling by Aberrant PI3K in Cancer / PIP3 activates AKT signaling / RAF/MAP kinase cascade / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / carbohydrate metabolic process / apical plasma membrane / extracellular space / extracellular exosome / extracellular region / membrane / plasma membrane
Similarity search - Function
Glycosyl hydrolases family 1, N-terminal conserved site / Glycosyl hydrolases family 1 N-terminal signature. / Glycosyl hydrolase family 1 / Glycoside hydrolase family 1 / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsSchnicker, N.J. / Xu, Z. / Mohammad, A. / Gakhar, L. / Huang, C.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)RO1 DK100605 United States
CitationJournal: Sci Rep / Year: 2025
Title: Conformational landscape of soluble α-klotho revealed by cryogenic electron microscopy.
Authors: Nicholas J Schnicker / Zhen Xu / Mohammad Amir / Lokesh Gakhar / Chou-Long Huang /
Abstract: α-Klotho (KLA) is a type-1 membranous protein that can associate with fibroblast growth factor receptor (FGFR) to form co-receptor for FGF23. The ectodomain of unassociated KLA is shed as soluble ...α-Klotho (KLA) is a type-1 membranous protein that can associate with fibroblast 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 flexible. Three-dimensional variability analysis revealed that apo-sKLA adopts conformations with different 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.
History
DepositionOct 3, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
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Revision 1.0Oct 16, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Klotho
B: Klotho


Theoretical massNumber of molelcules
Total (without water)232,6352
Polymers232,6352
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Klotho


Mass: 116317.477 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KL / Production host: Mus musculus (house mouse) / References: UniProt: Q9UEF7, beta-glucuronidase
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human aKlotho / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mus musculus (house mouse)
Buffer solutionpH: 7.5 / Details: 20mM HEPES pH7.5, 150mM NaCl, 1mM DTT
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
2150 mMNaCl1
31 mMDTT1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 279 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5760

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Processing

EM software
IDNameVersionCategory
4cryoSPARC3.3.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7891498
3D reconstructionResolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30219 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5w21

5w21


Pdb chain-ID: A / Accession code: 5w21 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00214504
ELECTRON MICROSCOPYf_angle_d0.64519736
ELECTRON MICROSCOPYf_dihedral_angle_d15.1995198
ELECTRON MICROSCOPYf_chiral_restr0.0412022
ELECTRON MICROSCOPYf_plane_restr0.0052552

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