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- PDB-7nf7: Ovine rBAT ectodomain homodimer, asymmetric unit -

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Basic information

Entry
Database: PDB / ID: 7nf7
TitleOvine rBAT ectodomain homodimer, asymmetric unit
Componentsneutral and basic amino acid transport protein rBAT
KeywordsTRANSLOCASE / Transporter / Cystinuria / Complex / Ectodomain
Function / homologyOligo-1,6-glucosidase, domain 2 / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Glycosyl hydrolase, all-beta / Glycoside hydrolase superfamily / carbohydrate metabolic process / Glycosyl hydrolase family 13 catalytic domain-containing protein
Function and homology information
Biological speciesOvis aries (sheep)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsLee, Y. / Kuehlbrandt, W.
Funding support Germany, 2items
OrganizationGrant numberCountry
Max Planck Society Germany
Human Frontier Science Program (HFSP)
Citation
Journal: Nat Commun / Year: 2022
Title: Ca-mediated higher-order assembly of heterodimers in amino acid transport system b biogenesis and cystinuria.
Authors: Yongchan Lee / Pattama Wiriyasermkul / Pornparn Kongpracha / Satomi Moriyama / Deryck J Mills / Werner Kühlbrandt / Shushi Nagamori /
Abstract: Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT ...Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter bAT, which constitute system b, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca mediates higher-order assembly of system b. Ca stabilizes the interface between two rBAT molecules, leading to super-dimerization of bAT-rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases.
#1: Journal: Biorxiv / Year: 2021
Title: Ca 2+ -mediated higher-order assembly of b 0,+ AT-rBAT is a key step for system b 0,+ biogenesis and cystinuria
Authors: Lee, Y. / Wiriyasermkul, P. / Moriyama, S. / Mills, D.J. / Kuhlbrandt, W. / Nagamori, S.
History
DepositionFeb 5, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 19, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 1, 2022Group: Database references / Derived calculations
Category: citation / citation_author / pdbx_struct_oper_list
Item: _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation

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Assembly

Deposited unit
A: neutral and basic amino acid transport protein rBAT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)80,5288
Polymers78,7541
Non-polymers1,7747
Water0
1
A: neutral and basic amino acid transport protein rBAT
hetero molecules

A: neutral and basic amino acid transport protein rBAT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,05616
Polymers157,5092
Non-polymers3,54714
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation1
Buried area1580 Å2
ΔGint6 kcal/mol
Surface area24730 Å2

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Components

#1: Protein neutral and basic amino acid transport protein rBAT


Mass: 78754.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ovis aries (sheep) / Gene: SLC3A1 / Production host: Homo sapiens (human) / References: UniProt: A0A6P7DVK7
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY

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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: Ovine rBAT ectodomain homodimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 79 kDa/nm / Experimental value: NO
Source (natural)Organism: Ovis aries (sheep)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_3689: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 581697 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0054949
ELECTRON MICROSCOPYf_angle_d0.5986717
ELECTRON MICROSCOPYf_dihedral_angle_d28.376680
ELECTRON MICROSCOPYf_chiral_restr0.044723
ELECTRON MICROSCOPYf_plane_restr0.005857

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