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- PDB-8oj4: Structure of the MlaCD complex (1:6 stoichiometry) -

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Basic information

Entry
Database: PDB / ID: 8oj4
TitleStructure of the MlaCD complex (1:6 stoichiometry)
Components
  • Intermembrane phospholipid transport system binding protein MlaC
  • Intermembrane phospholipid transport system binding protein MlaD
KeywordsLIPID BINDING PROTEIN / Outer membrane / gram-negative bacteria / lipid transfer / antibiotic resistance
Function / homology
Function and homology information


phospholipid transfer activity / intermembrane phospholipid transfer / phospholipid transporter activity / phospholipid-translocating ATPase complex / phospholipid transport / phospholipid binding / outer membrane-bounded periplasmic space / membrane / plasma membrane
Similarity search - Function
Tgt2/MlaC superfamily / Toluene tolerance Ttg2/phospholipid-binding protein MlaC / MlaC protein / Probable phospholipid ABC transporter-binding protein MlaD / : / Mce/MlaD / MlaD protein
Similarity search - Domain/homology
Intermembrane phospholipid transport system binding protein MlaC / Intermembrane phospholipid transport system binding protein MlaD
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.35 Å
AuthorsWotherspoon, P. / Bui, S. / Sridhar, P. / Bergeron, J.R.C. / Knowles, T.J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R019061/2 United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport.
Authors: Peter Wotherspoon / Hannah Johnston / David J Hardy / Rachel Holyfield / Soi Bui / Giedrė Ratkevičiūtė / Pooja Sridhar / Jonathan Colburn / Charlotte B Wilson / Adam Colyer / Benjamin F ...Authors: Peter Wotherspoon / Hannah Johnston / David J Hardy / Rachel Holyfield / Soi Bui / Giedrė Ratkevičiūtė / Pooja Sridhar / Jonathan Colburn / Charlotte B Wilson / Adam Colyer / Benjamin F Cooper / Jack A Bryant / Gareth W Hughes / Phillip J Stansfeld / Julien R C Bergeron / Timothy J Knowles /
Abstract: The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane ...The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.
History
DepositionMar 23, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2024Group: Data collection / Database references ...Data collection / Database references / Source and taxonomy / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / entity / entity_src_gen / struct_ref / struct_ref_seq
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 / _entity.pdbx_description / _entity_src_gen.pdbx_gene_src_gene / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Intermembrane phospholipid transport system binding protein MlaC
A: Intermembrane phospholipid transport system binding protein MlaD
B: Intermembrane phospholipid transport system binding protein MlaD
C: Intermembrane phospholipid transport system binding protein MlaD
D: Intermembrane phospholipid transport system binding protein MlaD
E: Intermembrane phospholipid transport system binding protein MlaD
F: Intermembrane phospholipid transport system binding protein MlaD


Theoretical massNumber of molelcules
Total (without water)141,5487
Polymers141,5487
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Intermembrane phospholipid transport system binding protein MlaC


Mass: 23989.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: mlaC, yrbC, b3192, JW3159 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ADV7
#2: Protein
Intermembrane phospholipid transport system binding protein MlaD


Mass: 19593.133 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: mlaD, yrbD, b3193, JW3160 / Production host: Escherichia coli (E. coli) / References: UniProt: P64604

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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: MlaCD / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18rc3_3805: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97460 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037110
ELECTRON MICROSCOPYf_angle_d0.5949681
ELECTRON MICROSCOPYf_dihedral_angle_d4.706988
ELECTRON MICROSCOPYf_chiral_restr0.0451140
ELECTRON MICROSCOPYf_plane_restr0.0051250

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