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- PDB-8gk7: MsbA bound to cerastecin C -

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Basic information

Entry
Database: PDB / ID: 8gk7
TitleMsbA bound to cerastecin C
ComponentsLipid A export ATP-binding/permease protein MsbA
KeywordsANTIMICROBIAL PROTEIN / MsbA / antibacterial / inhibitor / cerastecin
Function / homology
Function and homology information


ATPase-coupled lipid transmembrane transporter activity / ABC-type transporter activity / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
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 / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain ...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 / 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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / : / Lipid A export ATP-binding/permease protein MsbA
Similarity search - Component
Biological speciesAcinetobacter baumannii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsChen, Y. / Klein, D.
Funding support1items
OrganizationGrant numberCountry
Other private
CitationJournal: Nat Microbiol / Year: 2024
Title: Cerastecins inhibit membrane lipooligosaccharide transport in drug-resistant Acinetobacter baumannii.
Authors: Hao Wang / Andrii Ishchenko / Jason Skudlarek / Pamela Shen / Liudmila Dzhekieva / Ronald E Painter / Yun-Ting Chen / Marina Bukhtiyarova / Andrew Leithead / Rodger Tracy / Kerim Babaoglu / ...Authors: Hao Wang / Andrii Ishchenko / Jason Skudlarek / Pamela Shen / Liudmila Dzhekieva / Ronald E Painter / Yun-Ting Chen / Marina Bukhtiyarova / Andrew Leithead / Rodger Tracy / Kerim Babaoglu / Carolyn Bahnck-Teets / Alexei Buevich / Tamara D Cabalu / Marc Labroli / Henry Lange / Ying Lei / Wei Li / Jian Liu / Paul A Mann / Tao Meng / Helen J Mitchell / James Mulhearn / Giovanna Scapin / Deyou Sha / Anthony W Shaw / Qian Si / Ling Tong / Chengwei Wu / Zhe Wu / Jing Chen Xiao / Min Xu / Li-Kang Zhang / David McKenney / Randy R Miller / Todd A Black / Andrew Cooke / Carl J Balibar / Daniel J Klein / Izzat Raheem / Scott S Walker /
Abstract: Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of ...Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.
History
DepositionMar 17, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lipid A export ATP-binding/permease protein MsbA
B: Lipid A export ATP-binding/permease protein MsbA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,5785
Polymers133,7872
Non-polymers1,7913
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Lipid A export ATP-binding/permease protein MsbA


Mass: 66893.578 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acinetobacter baumannii (bacteria) / Gene: msbA / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A6F8TGG1
#2: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#3: Chemical ChemComp-ZQF / 2-[(4-butylbenzene-1-sulfonyl)amino]-5-[(3-{4-[(4-butylbenzene-1-sulfonyl)amino]-3-carboxyanilino}-3-oxopropyl)carbamoyl]benzoic acid


Mass: 778.891 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C38H42N4O10S2 / Feature type: SUBJECT OF INVESTIGATION
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: MsbA complexed with cerastecin C / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.45 MDa / Experimental value: YES
Source (natural)Organism: Acinetobacter baumannii (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 7
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 FIELDBright-field microscopy / Nominal defocus max: 4251 nm / Nominal defocus min: 1926 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 62.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1680369
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 296798 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028986
ELECTRON MICROSCOPYf_angle_d0.4212208
ELECTRON MICROSCOPYf_dihedral_angle_d4.5651336
ELECTRON MICROSCOPYf_chiral_restr0.0361438
ELECTRON MICROSCOPYf_plane_restr0.0021606

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