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- PDB-7oh5: Cryo-EM structure of Drs2p-Cdc50p in the E1-AlFx-ADP state -

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Basic information

Entry
Database: PDB / ID: 7oh5
TitleCryo-EM structure of Drs2p-Cdc50p in the E1-AlFx-ADP state
Components
  • Cell division control protein 50
  • Probable phospholipid-transporting ATPase DRS2,Probable phospholipid-transporting ATPase DRS2
KeywordsMEMBRANE PROTEIN / Lipid Flippase / P4 ATPase / trans-Golgi Network / Phosphatidylserine transport
Function / homology
Function and homology information


Cdc50p-Drs2p complex / Ion transport by P-type ATPases / phosphatidylcholine flippase activity / post-Golgi vesicle-mediated transport / phosphatidylserine flippase activity / ATPase-coupled intramembrane lipid transporter activity / phospholipid-translocating ATPase complex / phosphatidylserine floppase activity / phosphatidylethanolamine flippase activity / P-type phospholipid transporter ...Cdc50p-Drs2p complex / Ion transport by P-type ATPases / phosphatidylcholine flippase activity / post-Golgi vesicle-mediated transport / phosphatidylserine flippase activity / ATPase-coupled intramembrane lipid transporter activity / phospholipid-translocating ATPase complex / phosphatidylserine floppase activity / phosphatidylethanolamine flippase activity / P-type phospholipid transporter / endocytic recycling / phosphatidylinositol-4-phosphate binding / phospholipid translocation / membrane => GO:0016020 / Neutrophil degranulation / trans-Golgi network / endocytosis / endosome membrane / cell division / magnesium ion binding / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / Cation transport ATPase (P-type) / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site ...CDC50/LEM3 family / LEM3 (ligand-effect modulator 3) family / CDC50 family / P-type ATPase, subfamily IV / P-type ATPase, C-terminal / P-type ATPase, N-terminal / Phospholipid-translocating ATPase N-terminal / Phospholipid-translocating P-type ATPase C-terminal / Cation transport ATPase (P-type) / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
Chem-2Y5 / ADENOSINE-5'-DIPHOSPHATE / TETRAFLUOROALUMINATE ION / Cell division control protein 50 / Phospholipid-transporting ATPase DRS2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsTimcenko, M. / Dieudonne, T. / Montigny, C. / Boesen, T. / Lyons, J.A. / Lenoir, G. / Nissen, P.
Funding support Denmark, Germany, 3items
OrganizationGrant numberCountry
Lundbeckfonden Denmark
Novo Nordisk Foundation Denmark
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: J Mol Biol / Year: 2021
Title: Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase.
Authors: Milena Timcenko / Thibaud Dieudonné / Cédric Montigny / Thomas Boesen / Joseph A Lyons / Guillaume Lenoir / Poul Nissen /
Abstract: P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of ...P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na/K-ATPase or Ca-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.
History
DepositionMay 9, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 9, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entity_branch_descriptor / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: Probable phospholipid-transporting ATPase DRS2,Probable phospholipid-transporting ATPase DRS2
C: Cell division control protein 50
hetero molecules


Theoretical massNumber of molelcules
Total (without water)214,91211
Polymers211,5472
Non-polymers3,3659
Water362
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13010 Å2
ΔGint-51 kcal/mol
Surface area62500 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules AC

#1: Protein Probable phospholipid-transporting ATPase DRS2,Probable phospholipid-transporting ATPase DRS2


Mass: 164175.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Drs2p with a Thrombin cleavable C-terminal biotin acceptor domain (BAD) tag, and additional Thrombin cleavage site in the C-terminus.,Drs2p with a Thrombin cleavable C-terminal biotin ...Details: Drs2p with a Thrombin cleavable C-terminal biotin acceptor domain (BAD) tag, and additional Thrombin cleavage site in the C-terminus.,Drs2p with a Thrombin cleavable C-terminal biotin acceptor domain (BAD) tag, and additional Thrombin cleavage site in the C-terminus.
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: DRS2, YAL026C, FUN38 / Plasmid: pYeDP60 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): W303-1A dpep4
References: UniProt: P39524, P-type phospholipid transporter
#2: Protein Cell division control protein 50 / Y55_G0052250.mRNA.1.CDS.1


Mass: 47371.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Cdc50p with Thrombin cleavable C-terminal His-tag
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: PACBIOSEQ_LOCUS691, SCNYR20_0014012900 / Plasmid: pYeDP60 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): W303-1A dpep4 / References: UniProt: A0A6L0Z5H3

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Sugars , 3 types, 4 molecules

#3: 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
#4: Polysaccharide beta-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4) ...beta-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: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2-2/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][b-D-Manp]{}}}}}LINUCSPDB-CARE
#9: 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.: 1 / 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 , 5 types, 7 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-ALF / TETRAFLUOROALUMINATE ION


Mass: 102.975 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: AlF4
#7: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#8: Chemical ChemComp-2Y5 / (2R)-1-{[(R)-hydroxy{[(1R,2R,3R,4R,5S,6R)-2,3,5,6-tetrahydroxy-4-(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate / Phosphatidylinositol-4-phosphate / PI4P


Mass: 967.108 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H84O16P2
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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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

Component
IDNameTypeEntity IDParent-IDSource
1Binary complex of Drs2p-Cdc50p with the regulatory lipid PI4PCOMPLEX#1-#20RECOMBINANT
2Drs2pCOMPLEX#11RECOMBINANT
3Cdc50pCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)UnitsExperimental value
110.18 MDaNO
21MEGADALTONSNO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Saccharomyces cerevisiae (brewer's yeast)4932S288c
32Saccharomyces cerevisiae (brewer's yeast)4932S288c
43Saccharomyces cerevisiae (brewer's yeast)4932S288c
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Saccharomyces cerevisiae (brewer's yeast)4932W303-1A dpep4pYeDP60
32Saccharomyces cerevisiae (brewer's yeast)4932W303-1A dpep4pYeDP60
43Saccharomyces cerevisiae (brewer's yeast)4932W303-1A dpep4pYeDP60
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMMOPS-Tris1
2100 mMKCl1
35 mMMgCl21
41 mMDithiothreitolDTT1
50.03 mg/mLlauryl maltose neopentyl glycolLMNG1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified in detergent lauryl maltose neopentyl glycol (LMNG)
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 / Cs: 2.7 mm
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 6114
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18rc7_3834refinement
PHENIX1.18rc7_3834refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4cryoSPARC2CTF correctionpatch CTF
7Coot0.9-pre EL (ccpem)model fitting
9cryoSPARC2initial Euler assignmentheterogeneous refinement
10cryoSPARC2final Euler assignmentnon-uniform refinement
11cryoSPARC2classificationheterogeneous refinement
12cryoSPARC23D reconstructionnon-uniform refinement
13PHENIX1.18rc7-3834model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2423271
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 256001 / Symmetry type: POINT
Atomic model buildingB value: 62 / Space: REAL / Target criteria: correlation coefficient
Details: Domains were rigid body fit into the density and manually adjusted and reconnected.
Atomic model buildingPDB-ID: 6ROJ

6roj


Accession code: 6ROJ / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 72.16 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001811865
ELECTRON MICROSCOPYf_angle_d0.407816091
ELECTRON MICROSCOPYf_chiral_restr0.03921844
ELECTRON MICROSCOPYf_plane_restr0.00332003
ELECTRON MICROSCOPYf_dihedral_angle_d16.42074351

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