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- PDB-8pij: Neisseria meningitidis Type IV pilus SB-GATDH variant bound to th... -

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Basic information

Entry
Database: PDB / ID: 8pij
TitleNeisseria meningitidis Type IV pilus SB-GATDH variant bound to the C24 nanobody
Components
  • C24 nanobody
  • Pilin
KeywordsPROTEIN FIBRIL / Pilin / Extracellular / Adhesion / Aggregation
Function / homology
Function and homology information


pilus / cell adhesion / membrane
Similarity search - Function
Fimbrial protein pilin / Pilin (bacterial filament) / Prokaryotic N-terminal methylation motif / Prokaryotic N-terminal methylation site / Pilin-like
Similarity search - Domain/homology
SN-GLYCEROL-3-PHOSPHATE / : / Pilin
Similarity search - Component
Biological speciesNeisseria meningitidis 8013 (bacteria)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsFernandez-Martinez, D. / Dumenil, G.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR 18 CE11 0022 France
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms.
Authors: David Fernandez-Martinez / Youxin Kong / Sylvie Goussard / Agustin Zavala / Pauline Gastineau / Martial Rey / Gabriel Ayme / Julia Chamot-Rooke / Pierre Lafaye / Matthijn Vos / Ariel Mechaly ...Authors: David Fernandez-Martinez / Youxin Kong / Sylvie Goussard / Agustin Zavala / Pauline Gastineau / Martial Rey / Gabriel Ayme / Julia Chamot-Rooke / Pierre Lafaye / Matthijn Vos / Ariel Mechaly / Guillaume Duménil /
Abstract: Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade ...Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.
History
DepositionJun 21, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pilin
B: C24 nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,4434
Polymers30,9792
Non-polymers4642
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Pilin /


Mass: 17067.182 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis 8013 (bacteria) / Production host: Neisseria meningitidis 8013 (bacteria) / References: UniProt: A0A1I9GEU1
#2: Antibody C24 nanobody


Mass: 13911.585 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#3: Chemical ChemComp-G3P / SN-GLYCEROL-3-PHOSPHATE / Glycerol 3-phosphate


Mass: 172.074 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H9O6P / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-WKE / (2~{R})-~{N}-[(2~{R},3~{S},4~{S},5~{R},6~{R})-5-acetamido-2-methyl-4,6-bis(oxidanyl)oxan-3-yl]-2,3-bis(oxidanyl)propanamide


Mass: 292.286 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H20N2O7 / 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: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Complex of Neisseria meningitidis PilE SB-GATDH and the C24 nanobody
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Neisseria meningitidis 8013 (bacteria)
Source (recombinant)Organism: Neisseria meningitidis 8013 (bacteria)
Buffer solutionpH: 7.4
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmerty
IDImage processing-IDAngular rotation/subunit (°)Axial rise/subunit (Å)Axial symmetry
11100.75310.429C1
21100.75310.429C1
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 434690 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0012226
ELECTRON MICROSCOPYf_angle_d0.3753019
ELECTRON MICROSCOPYf_dihedral_angle_d3.065328
ELECTRON MICROSCOPYf_chiral_restr0.04349
ELECTRON MICROSCOPYf_plane_restr0.003383

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