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- PDB-8ufh: Acinetobacter baylyi LptB2FG bound to Acinetobacter baylyi lipopo... -

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Basic information

Entry
Database: PDB / ID: 8ufh
TitleAcinetobacter baylyi LptB2FG bound to Acinetobacter baylyi lipopolysaccharide and a macrocyclic peptide
Components
  • LPS export ABC transporter permease LptG
  • Lipopolysaccharide export system ATP-binding protein LptB
  • Lipopolysaccharide export system permease protein LptF
KeywordsLIPID TRANSPORT / lipopolysaccharide / ABC / ATPase / antibiotic / macrocyclic peptide / gram-negative bacteria / ESKAPE
Function / homology
Function and homology information


lipopolysaccharide transport / ATP-binding cassette (ABC) transporter complex / transmembrane transport / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Permease LptG/LptF-related / LPS export ABC transporter permease LptF / LPS export ABC transporter permease LptG / Lipopolysaccharide export system permease LptF/LptG / Branched-chain amino acid ATP-binding cassette transporter, C-terminal / Branched-chain amino acid ATP-binding cassette transporter / LPS export ABC transporter, ATP-binding protein LptB / : / ABC transporter-like, conserved site / ABC transporters family signature. ...Permease LptG/LptF-related / LPS export ABC transporter permease LptF / LPS export ABC transporter permease LptG / Lipopolysaccharide export system permease LptF/LptG / Branched-chain amino acid ATP-binding cassette transporter, C-terminal / Branched-chain amino acid ATP-binding cassette transporter / LPS export ABC transporter, ATP-binding protein LptB / : / 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
Chem-WJW / Chem-Y75 / Lipopolysaccharide export system ATP-binding protein LptB / Permease / Lipopolysaccharide export system permease protein LptF
Similarity search - Component
Biological speciesAcinetobacter baylyi ADP1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPahil, K.S. / Gilman, M.S.A. / Baidin, V. / Kruse, A.C. / Kahne, D.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI149778 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI081059 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI153358 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI158028 United States
Howard Hughes Medical Institute (HHMI)Hanna Gray Fellowship United States
CitationJournal: Nature / Year: 2024
Title: A new antibiotic traps lipopolysaccharide in its intermembrane transporter.
Authors: Karanbir S Pahil / Morgan S A Gilman / Vadim Baidin / Thomas Clairfeuille / Patrizio Mattei / Christoph Bieniossek / Fabian Dey / Dieter Muri / Remo Baettig / Michael Lobritz / Kenneth ...Authors: Karanbir S Pahil / Morgan S A Gilman / Vadim Baidin / Thomas Clairfeuille / Patrizio Mattei / Christoph Bieniossek / Fabian Dey / Dieter Muri / Remo Baettig / Michael Lobritz / Kenneth Bradley / Andrew C Kruse / Daniel Kahne /
Abstract: Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature ...Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.
History
DepositionOct 4, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 3, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jan 31, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lipopolysaccharide export system ATP-binding protein LptB
B: Lipopolysaccharide export system ATP-binding protein LptB
F: Lipopolysaccharide export system permease protein LptF
G: LPS export ABC transporter permease LptG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,0456
Polymers139,7104
Non-polymers3,3352
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Lipopolysaccharide export system ATP-binding protein LptB


Mass: 29032.344 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baylyi ADP1 (bacteria) / Gene: ACIAD1486 / Production host: Acinetobacter baylyi ADP1 (bacteria) / References: UniProt: Q6FC66
#2: Protein Lipopolysaccharide export system permease protein LptF


Mass: 41564.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baylyi ADP1 (bacteria) / Gene: ACIAD0254 / Production host: Acinetobacter baylyi ADP1 (bacteria) / References: UniProt: Q6FFD7
#3: Protein LPS export ABC transporter permease LptG


Mass: 40080.621 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baylyi ADP1 (bacteria) / Gene: ACIAD0255 / Production host: Acinetobacter baylyi ADP1 (bacteria) / References: UniProt: Q6FFD6
#4: Chemical ChemComp-WJW / (2~{R},4~{R},5~{R},6~{R})-2-[(2~{R},4~{R},5~{R},6~{R})-5-[(2~{S},4~{R},5~{R},6~{R})-4-[(2~{R},3~{R},4~{R},5~{S},6~{S})-3-acetamido-6-carboxy-4,5-bis(oxidanyl)oxan-2-yl]oxy-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-carboxy-5-oxidanyl-oxan-2-yl]oxy-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-2-carboxy-2-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-4-[(3~{S})-3-dodecanoyloxydodecanoyl]oxy-6-[[(2~{R},3~{S},4~{R},5~{R},6~{R})-5-[[(3~{R})-3-heptanoyloxynonanoyl]amino]-3-oxidanyl-4-[(3~{R})-3-oxidanyloctanoyl]oxy-6-phosphonooxy-oxan-2-yl]methoxy]-5-[[(3~{S})-3-[(3~{R})-3-oxidanyldodecanoyl]oxydecanoyl]amino]-3-phosphonooxy-oxan-2-yl]methoxy]oxan-4-yl]oxy-6-[(1~{R})-1,2-bis(oxidanyl)ethyl]-4,5-bis(oxidanyl)oxane-2-carboxylic acid


Mass: 2537.735 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C114H199N3O54P2
#5: Chemical ChemComp-Y75 / (7S,10S,13S,17P)-10-(4-aminobutyl)-7-(3-aminopropyl)-17-(6-aminopyridin-3-yl)-20-chloro-13-[(1H-indol-3-yl)methyl]-12-methyl-6,7,9,10,12,13,15,16-octahydropyrido[2,3-b][1,5,8,11,14]benzothiatetraazacycloheptadecine-8,11,14(5H)-trione


Mass: 797.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H49ClN10O3S
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: Inner membrane lipopolysaccharide transporter composed of LptF, LptG, and two molecules of LptB in complex with lipopolysaccharide and a macrocyclic peptide
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.139483 MDa / Experimental value: NO
Source (natural)Organism: Acinetobacter baylyi ADP1 (bacteria)
Source (recombinant)Organism: Acinetobacter baylyi ADP1 (bacteria) / Strain: C43
Buffer solutionpH: 7.4
Details: 300 mM NaCl, 20 mM Tris [pH 7.4], 0.02% GDN, 0.25 mM tris(hydroxypropyl)phosphine, 8 mM imidazole
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMSodium chlorideNaCl1
220 mMTris pH 7.4NH2C(CH2OH)31
30.02 %glyco-diosgeninC56H92O251
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse as determined by SEC.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 defocus max: 2400 nm / Nominal defocus min: 1100 nm
Image recordingElectron dose: 57.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1900000
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 503695 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039311
ELECTRON MICROSCOPYf_angle_d0.64412590
ELECTRON MICROSCOPYf_dihedral_angle_d4.6861417
ELECTRON MICROSCOPYf_chiral_restr0.0951439
ELECTRON MICROSCOPYf_plane_restr0.0041583

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