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- PDB-8p2x: Structure of human SIT1:ACE2 complex (open PD conformation) -

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Basic information

Entry
Database: PDB / ID: 8p2x
TitleStructure of human SIT1:ACE2 complex (open PD conformation)
Components
  • Processed angiotensin-converting enzyme 2
  • Sodium- and chloride-dependent transporter XTRP3
KeywordsMEMBRANE PROTEIN / COVID-19 receptor / amino acid transport
Function / homology
Function and homology information


proline:sodium symporter activity / L-isoleucine transmembrane transporter activity / solute:sodium symporter activity / L-isoleucine import across plasma membrane / L-proline import across plasma membrane / Variant SLC6A20 contributes towards hyperglycinuria (HG) and iminoglycinuria (IG) / Variant SLC6A20 contributes towards hyperglycinuria (HG) and iminoglycinuria (IG) / amino-acid betaine transport / proline import across plasma membrane / L-proline transmembrane transporter activity ...proline:sodium symporter activity / L-isoleucine transmembrane transporter activity / solute:sodium symporter activity / L-isoleucine import across plasma membrane / L-proline import across plasma membrane / Variant SLC6A20 contributes towards hyperglycinuria (HG) and iminoglycinuria (IG) / Variant SLC6A20 contributes towards hyperglycinuria (HG) and iminoglycinuria (IG) / amino-acid betaine transport / proline import across plasma membrane / L-proline transmembrane transporter activity / amino-acid betaine transmembrane transporter activity / glycine import across plasma membrane / glycine transport / proline transport / amino acid import across plasma membrane / neutral L-amino acid transmembrane transporter activity / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / Na+/Cl- dependent neurotransmitter transporters / amino acid transport / positive regulation of amino acid transport / angiotensin-converting enzyme 2 / positive regulation of L-proline import across plasma membrane / angiotensin-mediated drinking behavior / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / regulation of systemic arterial blood pressure by renin-angiotensin / positive regulation of gap junction assembly / tryptophan transport / regulation of cardiac conduction / regulation of vasoconstriction / peptidyl-dipeptidase activity / maternal process involved in female pregnancy / transporter activator activity / transport across blood-brain barrier / Metabolism of Angiotensinogen to Angiotensins / carboxypeptidase activity / angiotensin maturation / Attachment and Entry / receptor-mediated endocytosis of virus by host cell / metallocarboxypeptidase activity / viral life cycle / positive regulation of cardiac muscle contraction / sodium ion transmembrane transport / regulation of cytokine production / blood vessel diameter maintenance / negative regulation of smooth muscle cell proliferation / brush border membrane / negative regulation of ERK1 and ERK2 cascade / positive regulation of reactive oxygen species metabolic process / metallopeptidase activity / endocytic vesicle membrane / regulation of cell population proliferation / virus receptor activity / regulation of inflammatory response / endopeptidase activity / viral translation / Potential therapeutics for SARS / Induction of Cell-Cell Fusion / membrane fusion / entry receptor-mediated virion attachment to host cell / Attachment and Entry / receptor-mediated virion attachment to host cell / cilium / apical plasma membrane / membrane raft / endoplasmic reticulum lumen / symbiont entry into host cell / cell surface / negative regulation of transcription by RNA polymerase II / extracellular space / extracellular exosome / extracellular region / zinc ion binding / identical protein binding / membrane / plasma membrane
Similarity search - Function
Neutral amino acid SLC6 transporter / Sodium:neurotransmitter symporter family signature 2. / Sodium:neurotransmitter symporter family signature 1. / Sodium:neurotransmitter symporter / Sodium:neurotransmitter symporter superfamily / Sodium:neurotransmitter symporter family / Sodium:neurotransmitter symporter family profile. / Collectrin domain / Renal amino acid transporter / Collectrin-like domain profile. ...Neutral amino acid SLC6 transporter / Sodium:neurotransmitter symporter family signature 2. / Sodium:neurotransmitter symporter family signature 1. / Sodium:neurotransmitter symporter / Sodium:neurotransmitter symporter superfamily / Sodium:neurotransmitter symporter family / Sodium:neurotransmitter symporter family profile. / Collectrin domain / Renal amino acid transporter / Collectrin-like domain profile. / Peptidase M2, peptidyl-dipeptidase A / Angiotensin-converting enzyme / Peptidase family M2 domain profile. / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
2-acetamido-2-deoxy-alpha-D-glucopyranose / Angiotensin-converting enzyme 2 / Sodium- and chloride-dependent transporter XTRP3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsLi, H.Z. / Pike, A.C.W. / Chi, G. / Hansen, J.S. / Lee, S.G. / Rodstrom, K.E.J. / Bushell, S.R. / Speedman, D. / Evans, A. / Wang, D. ...Li, H.Z. / Pike, A.C.W. / Chi, G. / Hansen, J.S. / Lee, S.G. / Rodstrom, K.E.J. / Bushell, S.R. / Speedman, D. / Evans, A. / Wang, D. / He, D. / Shrestha, L. / Nasrallah, C. / Chalk, R. / Moreira, T. / MacLean, E.M. / Marsden, B. / Bountra, C. / Burgess-Brown, N.A. / Dafforn, T.R. / Carpenter, E.P. / Sauer, D.B.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI) United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2.
Authors: Huanyu Z Li / Ashley C W Pike / Irina Lotsaris / Gamma Chi / Jesper S Hansen / Sarah C Lee / Karin E J Rödström / Simon R Bushell / David Speedman / Adam Evans / Dong Wang / Didi He / ...Authors: Huanyu Z Li / Ashley C W Pike / Irina Lotsaris / Gamma Chi / Jesper S Hansen / Sarah C Lee / Karin E J Rödström / Simon R Bushell / David Speedman / Adam Evans / Dong Wang / Didi He / Leela Shrestha / Chady Nasrallah / Nicola A Burgess-Brown / Robert J Vandenberg / Timothy R Dafforn / Elisabeth P Carpenter / David B Sauer /
Abstract: Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and ...Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.
History
DepositionMay 16, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 12, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Processed angiotensin-converting enzyme 2
B: Processed angiotensin-converting enzyme 2
C: Sodium- and chloride-dependent transporter XTRP3
D: Sodium- and chloride-dependent transporter XTRP3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)335,26620
Polymers329,6044
Non-polymers5,66216
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_1ens_2chain "C"
d_2ens_2chain "D"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1THRLYSA1 - 750
d_12ens_1ZNZNA
d_13ens_1NAGNAGA
d_14ens_1NAGNAGA
d_15ens_1BMABMAA
d_16ens_1NDGNDGA
d_17ens_1NAGNAGA
d_18ens_1NAGNAGA
d_19ens_1NAGNAGA
d_110ens_1NAGNAGA
d_111ens_1MANMANA
d_112ens_1MANMANA
d_113ens_1MANMANA
d_114ens_1MANMANA
d_21ens_1THRLYSB1 - 750
d_22ens_1ZNZNB
d_23ens_1NAGNAGB
d_24ens_1NAGNAGB
d_25ens_1BMABMAB
d_26ens_1NDGNDGB
d_27ens_1NAGNAGB
d_28ens_1NAGNAGB
d_29ens_1NAGNAGB
d_210ens_1NAGNAGB
d_211ens_1MANMANB
d_212ens_1MANMANB
d_213ens_1MANMANB
d_214ens_1MANMANB
d_11ens_2SERARGC1 - 572
d_12ens_2NAGNAGC
d_13ens_2NAGNAGC
d_21ens_2SERARGD1 - 572
d_22ens_2NAGNAGD
d_23ens_2NAGNAGD

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(-0.999998832128, 0.00134090031451, -0.000733300372691), (-0.00133744942468, -0.999988124442, -0.00468638499728), (-0.000739575639456, -0.00468539877201, 0.99998874997)319.780017911, 321.05587777, 0.814053560843
2given(-0.999958126522, 0.00758776502676, -0.00511576237603), (-0.00760972314615, -0.999961858146, 0.00428653319549), (-0.0050830420447, 0.0043252832388, 0.999977727056)319.510226079, 320.315565358, 0.289935162424

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Components

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Protein , 2 types, 4 molecules ABCD

#1: Protein Processed angiotensin-converting enzyme 2


Mass: 93425.570 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACE2, UNQ868/PRO1885 / Production host: Homo sapiens (human) / References: UniProt: Q9BYF1
#2: Protein Sodium- and chloride-dependent transporter XTRP3 / Sodium/imino-acid transporter 1 / Solute carrier family 6 member 20 / Transporter rB21A homolog


Mass: 71376.258 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A20, SIT1, XT3, XTRP3 / Plasmid: pHTBV1.1-CT10H-SIII-LIC / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q9NP91

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Sugars , 4 types, 14 molecules

#3: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]alpha-D-mannopyranose-(1-6)-[alpha-D- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1235.105 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpa1-6[DManpa1-3]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,7,6/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3-3/a4-b1_b4-c1_c3-d1_c6-e1_e3-f1_e6-g1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}}LINUCSPDB-CARE
#4: 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-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Sugar ChemComp-NDG / 2-acetamido-2-deoxy-alpha-D-glucopyranose / N-acetyl-alpha-D-glucosamine / 2-acetamido-2-deoxy-alpha-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / 2-(ACETYLAMINO)-2-DEOXY-A-D-GLUCOPYRANOSE


Type: D-saccharide, alpha linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-a-D-glucopyranosamineCOMMON NAMEGMML 1.0
a-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#7: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 8 / 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

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Non-polymers , 1 types, 2 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationY

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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: SIT1:ACE2 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.34 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES1
2150 mMsodium chloride1
30.02 w/vglyco-diosgenin (GDN)1
410 mMglycine1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.5 K / Details: grid blotted for 3sec

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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 defocus max: 2200 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 50.09 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8704
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 5 eV
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2SerialEMimage acquisition
4cryoSPARC3.3.1CTF correction
7Coot0.9.6model fitting
9cryoSPARC3.3.1initial Euler assignment
10cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.13D reconstruction
13PHENIX1.20_4459model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 509681
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 136985 / Algorithm: FOURIER SPACE / Details: cryoSPARC Non-uniform Refinement (C2 symmetry) / Symmetry type: POINT
Atomic model buildingB value: 147 / Protocol: FLEXIBLE FIT / Space: REAL
Details: Initial model fitting was peformed in Chimera and model building in COOT
Atomic model buildingPDB-ID: 6M1D

6m1d


Pdb chain-ID: D / Accession code: 6M1D / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 120.83 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001821392
ELECTRON MICROSCOPYf_angle_d0.408529198
ELECTRON MICROSCOPYf_chiral_restr0.03793316
ELECTRON MICROSCOPYf_plane_restr0.00313670
ELECTRON MICROSCOPYf_dihedral_angle_d9.51977436
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2BELECTRON MICROSCOPYNCS constraints6.19695986461E-13
ens_2d_2DELECTRON MICROSCOPYNCS constraints2.90126241849E-13

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