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- PDB-8v3j: Structure-Based Engineering of a Highly Immunogenic, Conformation... -

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Basic information

Entry
Database: PDB / ID: 8v3j
TitleStructure-Based Engineering of a Highly Immunogenic, Conformationally Stabilized FimH Antigen for a Urinary Tract Infection Vaccine
ComponentsType 1 fimbrin D-mannose specific adhesin,Protein FimG
KeywordsCELL ADHESION / TYPE I PILUS / CATCH-BOND / LECTIN / UPEC / BACTERIAL ADHESIN / UTI / MANNOSE
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 speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.898 Å
AuthorsJasti, J.
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
DepositionNov 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release
Revision 1.1Mar 12, 2025Group: Database references / Category: citation / citation_author
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type 1 fimbrin D-mannose specific adhesin,Protein FimG
B: Type 1 fimbrin D-mannose specific adhesin,Protein FimG
C: Type 1 fimbrin D-mannose specific adhesin,Protein FimG
D: Type 1 fimbrin D-mannose specific adhesin,Protein FimG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)129,3598
Polymers128,4744
Non-polymers8854
Water15,439857
1
A: Type 1 fimbrin D-mannose specific adhesin,Protein FimG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,3402
Polymers32,1191
Non-polymers2211
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Type 1 fimbrin D-mannose specific adhesin,Protein FimG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,3402
Polymers32,1191
Non-polymers2211
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
C: Type 1 fimbrin D-mannose specific adhesin,Protein FimG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,3402
Polymers32,1191
Non-polymers2211
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
4
D: Type 1 fimbrin D-mannose specific adhesin,Protein FimG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,3402
Polymers32,1191
Non-polymers2211
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)140.040, 149.260, 99.350
Angle α, β, γ (deg.)90.00, 130.03, 90.00
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-772-

HOH

21B-675-

HOH

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Components

#1: Protein
Type 1 fimbrin D-mannose specific adhesin,Protein FimG / Protein FimH


Mass: 32118.596 Da / Num. of mol.: 4 / Mutation: N7S,G15A,G16A,V27A,N70S,N228Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: fimH, b4320, JW4283, fimG, b4319, JW4282 / Cell line (production host): Expi293 cells / Production host: Homo sapiens (human) / References: UniProt: P08191, UniProt: P08190
#2: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 857 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.09 Å3/Da / Density % sol: 60.25 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 4.5 / Details: 0.1 M Sodium Acetate (pH 4.5), 25% (w/v) PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 17-ID / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Sep 24, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.898→87.086 Å / Num. obs: 86513 / % possible obs: 91 % / Redundancy: 3.5 % / CC1/2: 0.998 / Net I/σ(I): 15.1
Reflection shellResolution: 2.161→2.168 Å / Redundancy: 2.9 % / Num. unique obs: 829 / CC1/2: 0.739 / % possible all: 97.8

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Processing

Software
NameVersionClassification
BUSTER2.11.8 (22-FEB-2023)refinement
autoPROCdata reduction
autoPROCdata scaling
BUSTERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.898→27.5 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.929 / SU R Cruickshank DPI: 0.189 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.195 / SU Rfree Blow DPI: 0.165 / SU Rfree Cruickshank DPI: 0.164
RfactorNum. reflection% reflectionSelection details
Rfree0.2384 4469 5.17 %RANDOM
Rwork0.2109 ---
obs0.2124 86478 70.4 %-
Displacement parametersBiso mean: 41.68 Å2
Baniso -1Baniso -2Baniso -3
1-0.8766 Å20 Å20.157 Å2
2---0.656 Å20 Å2
3----0.2206 Å2
Refine analyzeLuzzati coordinate error obs: 0.3 Å
Refinement stepCycle: LAST / Resolution: 1.898→27.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8026 0 56 858 8940
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0088275HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.9911367HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d2580SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes1416HARMONIC5
X-RAY DIFFRACTIONt_it8275HARMONIC10
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_omega_torsion3.58
X-RAY DIFFRACTIONt_other_torsion16.38
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion1181SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact6980SEMIHARMONIC4
LS refinement shellResolution: 1.9→1.99 Å / Total num. of bins used: 51
RfactorNum. reflection% reflection
Rfree0.3028 -4.45 %
Rwork0.3042 1653 -
all0.3042 1730 -
obs--10.5 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.166-0.28310.00210.8363-0.53930.3304-0.0271-0.05260.02880.0085-0.0221-0.0239-0.0118-0.00360.04920.01950.0105-0.0132-0.0316-0.0011-0.0174-21.192231.497224.3869
20.1272-0.1461-0.01721.01660.55660.507-0.0215-0.0578-0.0177-0.0031-0.0122-0.0006-0.01270.0290.03370.00390.0013-0.019-0.0140.0186-0.040525.625424.463426.3485
30.58720.32940.45910.17580.13310.4296-0.0299-0.17660.03930.0569-0.0463-0.01350.03880.04350.0762-0.0547-0.00170.018-0.0074-0.0055-0.0598-3.8977.088124.7545
41.8221.1878-0.20461.27970.11930.43460.134-0.26810.2060.1767-0.16320.25970.0520.00460.02920.0087-0.04670.043-0.0225-0.05970.05714.023249.88720.5154
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1{ A|* }
2X-RAY DIFFRACTION2{ B|* }
3X-RAY DIFFRACTION3{ C|* }
4X-RAY DIFFRACTION4{ D|* }

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