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- PDB-8v93: Cryo-EM structure of E. coli FimH lectin domain bound to Fabs 329... -

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Basic information

Entry
Database: PDB / ID: 8v93
TitleCryo-EM structure of E. coli FimH lectin domain bound to Fabs 329-2 and 454-3
Components
  • Fab 329-2 heavy chain
  • Fab 329-2 light chain
  • Fab 454-3 heavy chain
  • Fab 454-3 light chain
  • Type 1 fimbrin D-mannose specific adhesin FimH, Donor strand complemented with FimG peptide 'triple mutant'
KeywordsCELL ADHESION/IMMUNE SYSTEM / Bacterial proteins / lectin domain / Fab / urinary tract infections / CELL ADHESION-IMMUNE SYSTEM complex
Function / homology
Function and homology information


pilus tip / mechanosensory behavior / cell adhesion involved in single-species biofilm formation / pilus / cell-substrate adhesion / D-mannose binding / host cell membrane / cell adhesion
Similarity search - Function
FimH, mannose-binding domain / FimH, mannose binding / Fimbrial-type adhesion domain / Fimbrial protein / : / Fimbrial-type adhesion domain superfamily / Adhesion domain superfamily
Similarity search - Domain/homology
Protein FimG / Type 1 fimbrin D-mannose specific adhesin
Similarity search - Component
Biological speciesMus musculus (house mouse)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsLees, J.A. / Han, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: PLoS Pathog / Year: 2025
Title: Structure-based design of an immunogenic, conformationally stabilized FimH antigen for a urinary tract infection vaccine.
Authors: Natalie C Silmon de Monerri / Ye Che / Joshua A Lees / Jayasankar Jasti / Huixian Wu / Matthew C Griffor / Srinivas Kodali / Julio Cesar Hawkins / Jacqueline Lypowy / Christopher Ponce / ...Authors: Natalie C Silmon de Monerri / Ye Che / Joshua A Lees / Jayasankar Jasti / Huixian Wu / Matthew C Griffor / Srinivas Kodali / Julio Cesar Hawkins / Jacqueline Lypowy / Christopher Ponce / Kieran Curley / Alexandre Esadze / Juan Carcamo / Thomas McLellan / David Keeney / Arthur Illenberger / Yury V Matsuka / Suman Shanker / Laurent Chorro / Alexey V Gribenko / Seungil Han / Annaliesa S Anderson / Robert G K Donald /
Abstract: Adhesion of E. coli to the urinary tract epithelium is a critical step in establishing urinary tract infections. FimH is an adhesin positioned on the fimbrial tip which binds to mannosylated proteins ...Adhesion of E. coli to the urinary tract epithelium is a critical step in establishing urinary tract infections. FimH is an adhesin positioned on the fimbrial tip which binds to mannosylated proteins on the urinary tract epithelium via its lectin domain (FimHLD). FimH is of interest as a target of vaccines to prevent urinary tract infections (UTI). Previously, difficulties in obtaining purified recombinant FimH from E. coli along with the poor inherent immunogenicity of FimH have hindered the development of effective FimH vaccine candidates. To overcome these challenges, we have devised a novel production method using mammalian cells to produce high yields of homogeneous FimH protein with comparable biochemical and immunogenic properties to FimH produced in E. coli. Next, to optimize conformational stability and immunogenicity of FimH, we used a computational approach to design improved FimH mutants and evaluated their biophysical and biochemical properties, and murine immunogenicity using a bacterial adhesion inhibition assay. This approach identified an immunogenic FimH variant (FimH-donor-strand complemented with FimG peptide 'triple mutant', FimH-DSG TM) capable of blocking bacterial adhesion that is produced at high yields in mammalian cells. By x-ray crystallography, we confirmed that the stabilized structure of the FimHLD in FimH-DSG TM is similar to native FimH on the fimbrial tip. Characterization of monoclonal antibodies elicited by FimH-DSG that can block bacterial binding to mannosylated surfaces identified 4 non-overlapping binding sites whose epitopes were mapped via a combinatorial cryogenic electron microscopy approach. Novel inhibitory epitopes in the lectin binding FimH were identified, revealing diverse functional mechanisms of FimH-directed antibodies with relevance to FimH-targeted UTI vaccines.
History
DepositionDec 7, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Fab 454-3 heavy chain
B: Fab 329-2 heavy chain
C: Fab 454-3 light chain
D: Type 1 fimbrin D-mannose specific adhesin FimH, Donor strand complemented with FimG peptide 'triple mutant'
L: Fab 329-2 light chain


Theoretical massNumber of molelcules
Total (without water)127,2875
Polymers127,2875
Non-polymers00
Water00
1


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

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Components

#1: Antibody Fab 454-3 heavy chain


Mass: 23940.627 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#2: Antibody Fab 329-2 heavy chain


Mass: 24103.885 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Antibody Fab 454-3 light chain


Mass: 23512.791 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#4: Protein Type 1 fimbrin D-mannose specific adhesin FimH, Donor strand complemented with FimG peptide 'triple mutant' / Protein FimH


Mass: 32118.596 Da / Num. of mol.: 1 / Mutation: N7S, G15A, G16A, V27A, N70S, N228Q within FimH
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: fimH, b4320, JW4283, fimG, b4319, JW4282 / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P08191, UniProt: P08190
#5: Antibody Fab 329-2 light chain


Mass: 23611.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
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

ComponentName: Ternary complex of E. coli FimH with Fabs 329-2 and 454-3
Type: COMPLEX / Entity ID: #2, #1, #5, #3-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
Buffer solutionpH: 7.5
SpecimenConc.: 0.25 mg/ml / Embedding 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: 2400 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 49 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80368 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044799
ELECTRON MICROSCOPYf_angle_d0.9446531
ELECTRON MICROSCOPYf_dihedral_angle_d6.133660
ELECTRON MICROSCOPYf_chiral_restr0.055724
ELECTRON MICROSCOPYf_plane_restr0.009838

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