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- PDB-8dhy: N-terminal fragment of MsbA fused to GFP in complex with copper(II) -

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Basic information

Entry
Database: PDB / ID: 8dhy
TitleN-terminal fragment of MsbA fused to GFP in complex with copper(II)
ComponentsFusion protein of MsbA N-terminal fragment and GFP,Green fluorescent protein
KeywordsFLUORESCENT PROTEIN / copper binding / MsbA
Function / homology
Function and homology information


MsbA transporter complex / lipopolysaccharide floppase activity / lipid translocation / ABC-type lipid A-core oligosaccharide transporter / lipopolysaccharide transport / ATPase-coupled lipid transmembrane transporter activity / ABC-type xenobiotic transporter activity / lipid transport / ATP-binding cassette (ABC) transporter complex / bioluminescence ...MsbA transporter complex / lipopolysaccharide floppase activity / lipid translocation / ABC-type lipid A-core oligosaccharide transporter / lipopolysaccharide transport / ATPase-coupled lipid transmembrane transporter activity / ABC-type xenobiotic transporter activity / lipid transport / ATP-binding cassette (ABC) transporter complex / bioluminescence / generation of precursor metabolites and energy / transmembrane transport / lipid binding / ATP hydrolysis activity / ATP binding / membrane / identical protein binding / plasma membrane
Similarity search - Function
Lipid A export ATP-binding/permease protein msbA family profile. / ABC transporter, lipid A-core flippase, MsbA / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein ...Lipid A export ATP-binding/permease protein msbA family profile. / ABC transporter, lipid A-core flippase, MsbA / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
COPPER (II) ION / Green fluorescent protein / ATP-dependent lipid A-core flippase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Aequorea victoria (jellyfish)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.15 Å
AuthorsSchrecke, S.R. / Zhang, T. / Lyu, J. / Laganowsky, A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM139876 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM138863 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for lipid and copper regulation of the ABC transporter MsbA.
Authors: Jixing Lyu / Chang Liu / Tianqi Zhang / Samantha Schrecke / Nicklaus P Elam / Charles Packianathan / Georg K A Hochberg / David Russell / Minglei Zhao / Arthur Laganowsky /
Abstract: A critical step in lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation ...A critical step in lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation carried out by the ATP-binding cassette transporter MsbA. Although LPS binding to the inner cavity of MsbA is well established, the selectivity of MsbA-lipid interactions at other site(s) remains poorly understood. Here we use native mass spectrometry (MS) to characterize MsbA-lipid interactions and guide structural studies. We show the transporter co-purifies with copper(II) and metal binding modulates protein-lipid interactions. A 2.15 Å resolution structure of an N-terminal region of MsbA in complex with copper(II) is presented, revealing a structure reminiscent of the GHK peptide, a high-affinity copper(II) chelator. Our results demonstrate conformation-dependent lipid binding affinities, particularly for the LPS-precursor, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)-lipid A (KDL). We report a 3.6 Å-resolution structure of MsbA trapped in an open, outward-facing conformation with adenosine 5'-diphosphate and vanadate, revealing a distinct KDL binding site, wherein the lipid forms extensive interactions with the transporter. Additional studies provide evidence that the exterior KDL binding site is conserved and a positive allosteric modulator of ATPase activity, serving as a feedforward activation mechanism to couple transporter activity with LPS biosynthesis.
History
DepositionJun 28, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 7, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.2Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Fusion protein of MsbA N-terminal fragment and GFP,Green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,2432
Polymers27,1801
Non-polymers641
Water81145
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, Native mass spectrometry shows fusion protein is a monomer and bound to one copper(II).
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)70.610, 108.540, 140.400
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number24
Space group name H-MI212121
Space group name HallI2b2c
Symmetry operation#1: x,y,z
#2: x,-y,-z+1/2
#3: -x+1/2,y,-z
#4: -x,-y+1/2,z
#5: x+1/2,y+1/2,z+1/2
#6: x+1/2,-y+1/2,-z+1
#7: -x+1,y+1/2,-z+1/2
#8: -x+1/2,-y+1,z+1/2

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Components

#1: Protein Fusion protein of MsbA N-terminal fragment and GFP,Green fluorescent protein /


Mass: 27179.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Aequorea victoria (jellyfish)
Gene: msbA, gfp / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P60752, UniProt: A0A059PIQ0, ABC-type lipid A-core oligosaccharide transporter
#2: Chemical ChemComp-CU / COPPER (II) ION / Copper


Mass: 63.546 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cu / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 45 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.95 Å3/Da / Density % sol: 75.15 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: 70% Tacsimate pH 7.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 1.3782 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Jun 7, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.3782 Å / Relative weight: 1
ReflectionResolution: 2.15→70 Å / Num. obs: 56321 / % possible obs: 99.3 % / Redundancy: 6.7 % / Biso Wilson estimate: 52.32 Å2 / CC1/2: 0.997 / Rrim(I) all: 0.094 / Net I/σ(I): 12.35
Reflection shellResolution: 2.15→2.21 Å / Redundancy: 6.8 % / Mean I/σ(I) obs: 1.61 / Num. unique obs: 4109 / CC1/2: 0.788 / Rrim(I) all: 1.45 / % possible all: 98

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
XDSJan 10, 2022data reduction
XSCALEJan 10, 2022data scaling
Coot0.9.6model building
PHENIX1.19.2_4158phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2B3P

2b3p


Resolution: 2.15→63.08 Å / SU ML: 0.2634 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.273
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2277 2830 5.03 %
Rwork0.2016 53401 -
obs0.2029 56231 99.12 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 59.36 Å2
Refinement stepCycle: LAST / Resolution: 2.15→63.08 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1887 0 1 45 1933
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01191938
X-RAY DIFFRACTIONf_angle_d1.16832624
X-RAY DIFFRACTIONf_chiral_restr0.0577283
X-RAY DIFFRACTIONf_plane_restr0.0134346
X-RAY DIFFRACTIONf_dihedral_angle_d16.9563708
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.15-2.190.35441360.32652624X-RAY DIFFRACTION97.91
2.19-2.230.2871400.29532677X-RAY DIFFRACTION97.88
2.23-2.270.35621440.29252667X-RAY DIFFRACTION98.08
2.27-2.320.37021390.31192625X-RAY DIFFRACTION98.29
2.32-2.370.27261380.26682612X-RAY DIFFRACTION98.53
2.37-2.420.30311390.26512654X-RAY DIFFRACTION98.66
2.42-2.480.26791400.24862661X-RAY DIFFRACTION99.15
2.48-2.550.27071450.23942693X-RAY DIFFRACTION98.99
2.55-2.620.33431420.2442634X-RAY DIFFRACTION98.58
2.62-2.710.26221440.25362695X-RAY DIFFRACTION99.16
2.71-2.810.29441410.27182678X-RAY DIFFRACTION99.16
2.81-2.920.30851410.30122661X-RAY DIFFRACTION99.5
2.92-3.050.32331400.26182675X-RAY DIFFRACTION99.72
3.05-3.210.21661450.22642699X-RAY DIFFRACTION99.75
3.21-3.410.25891430.21252709X-RAY DIFFRACTION99.96
3.41-3.680.20161480.19282686X-RAY DIFFRACTION99.82
3.68-4.050.22351450.17532670X-RAY DIFFRACTION99.89
4.05-4.630.16261360.14822703X-RAY DIFFRACTION100
4.63-5.830.18691440.14592681X-RAY DIFFRACTION99.65
5.84-63.080.18761400.17482697X-RAY DIFFRACTION99.79

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