Lieven ButsVrije Universiteit Brussel

Carbohydrate Recognition by the Fimbrial Adhesion Systems

of Escherichia coli191st Meeting of the Belgian Society

for Biochemistry and Molecular Biology2-Dec-2005

Protein-Carbohydrate BindingMediates Biological Recognition

● Carbohydrates are highly flexible molecules with a complex stereochemistry and a high density of functional groups; this makes them extremely suitable as information carriers

● The information is encoded by the genome in an indirect way, subject to complex regulation

● Lectins are proteins of non-immune origin that reversibly bind specific carbohydrate structures, without modifying the covalent structure of their ligands

● Biological recognition processes range from microbial infection over intracellular protein trafficking to the differentiation of cells and tissues in higher organisms

Carbohydrates as Labels

Blood stream

Site ofinfection

Carbohydrates Lectins

Rolling (weak adhesion)

L-selectin ligandsE-selectin ligands E-selectin

L-selectin

Strong adhesion and tissue invasion

Intact, healthy tissue

Carbohydrates as LiabilitiesInfluenzavirus particle

Respiratory tract epithelium

Haemagglutinin

Fimbriae or pili bear sugar-binding proteins (adhesins) along their length or at their tips and are involved in the attachment of the bacteria to substrates and host cells; they determine host range and tissue tropism

Bacterial Adhesins

Adhesion Systems

Chaperone/usherpathway

Nucleation/precipitationpathway

Neisseria meningitidis

Outer membraneadhesins

Moraxella catarrhalis

Adhesins of Gram-positive bacteria

General secretionpathway

Type 1 P-type F17F18 F4/K88 F5/K99

Escherichia coli, Salmonella, ...

Thin aggregative piliCurli

Type IV piliPseudomonas aeruginosaVibrio cholerae

Invasion systemsCS-1 AIDA

F17 Fimbriae

F17 operon: only 4 genes

Found in enterotoxicogenic Escherichia coli (ETEC) strains infecting livestock

outer membrane

Usher

C

Fimbria(flexible)

G

DChaperone

A

F17-G: adhesin with GlcNAc specificity

Pilin domain

binding site

C

N

The Adhesin has Two Domains

Complete Ig fold

C

Lectindomain

short linker

The Adhesin has Two Domains

C

Pilin domain

Lectindomain

short linker

binding site

C

N

Structuralpilin subunit

N-terminalextension

missing strand inincomplete Ig fold

The Adhesin has Two Domains

C

Pilin domain

Lectindomain

short linker

binding site

C

N

N-terminalextension

missing strand inincomplete Ig fold

The Adhesin has Two Domains

C

Pilin domain

Lectindomain

short linker

binding site

C

N

N-terminalextension

The Adhesin has Two Domains

C

Lectindomain

short linker

binding site

F17 Fimbriae

F17 operon: only 4 genes

Found in enterotoxicogenic Escherichia coli (ETEC) strains infecting livestock

outer membrane

Usher

C

Fimbria(flexible)

G

DChaperone

A

F17-G: adhesin with GlcNAc specificity

N

C

A Specific ChaperoneAssists Folding and Assembly

N

C

Donor strandcomplementation

Donor strandexchange

First subunit

Chaperone

G

NTESecondsubunit(truncated)

Zavialov et al., Cell, 113; 587-596.

An X-Ray Snapshot

A

Outer membrane

Usher

D

Pilus rod(rigid)

AdaptersFG

H FimH adhesin

C Chaperone

A

Type 1 Pili

fim operon

Found in human uropathogenic Escherichia coli (UPEC) strains

FimH adhesin has a basic specificity for mannose

Therapeutic Strategies

● General antibiotics

● Interfering with pilus assembly

● Interfering with adhesion

● Optimal approach depends on circumstances● Potential for complementarity

● Resistance can arise if used inappropriately● Persistent and recurrent infections can lead to

very long treatment times

● “Pilicides” specifically target chaperone function

● Carbohydrate-based binding site blockers target the adhesin-receptor interaction

F17G Exhibits SignificantNatural Variation

F17a-G, F17d-G: bovine enterotoxigenic E. coli (ETEC) strains

F17b-G: E. coli strains isolated from septicemic calves and lambs

F17c-G: associated with bovine diarrhea or septicemia and with

lambs showing nephrosis

F17e-G (E. coli strain CK210)

F17f-G (E. coli CK377 strain): expressed by non-enterotoxigenic

E. coli isolated from lambs and goat kids

G fimbriae from human uropathogenic E. coli strains were found

to be identical in amino acid sequence to F17c fimbriae

Crystal Structure of theF17a-G Lectin Domain

.

OHO

HONHAc

OH

OH

2 -acetamido-2-deoxy-D -glucopyranose

OHO

HONHAc

SeMe

OH

methyl 2-acetamido-2-deoxy-1-seleno- -D-glucopyranoside

(Oscarson lab)

Crystal Packing Variation

Sequence Variation

Crystal Quality Variation

Time to obtain crystals

Maximumresolution

Suitability forsoaking withnew ligands

F17bGF17cG

F17dG

F17eGF17fG

F17aGDays

Minutes

No crystalsobtained N/A N/A

1.1 Å2.1 Å1.8 Å

2.4 Å1.5 Å

+++-

--

-

Days

DaysDays

Prokaryotic genetic diversity is a sourceof complications and opportunities

Carbohydrate Complexes

GlcNAc(1-3)Gal -methyl-paranitro-phenyl-GlcNAc

Surface Plasmon ResonanceBinding Experiments

Surface Plasmon ResonanceBinding Experiments

Glcα1-OMe

Glcβ1-OMe

Time

Response(Resonance

Units)

F17a-G F17d-GNAG 0.85 ± 0.09 1.00 ± 0.21

1.09 ± 0.04 0.97 ± 0.08

1.02 ± 0.08 1.33 ± 0.11

1.62 ± 0.08 1.92 ± 0.114.58 ± 0.17 4.59 ± 0.562.78 ± 0.10 2.99 ± 0.14

tri 0.64 ± 0.05 0.55 ± 0.08penta 1.19 ± 0.04 1.24 ± 0.05GlcN < 0.1 < 0.1

Not detectable Not detectable< 0.1 < 0.1

Gal Not detectable Not detectableGalNAc Not detectable Not detectable

Ka (1/mM)

NAG2

NAG3

NAG4

β-Me-SeNAGNAGβ1-2Man

α-Me-Glcβ-Me-Glc

Surface Plasmon ResonanceBinding Experiments

Type 1 Pili

A

Outer membrane

Usher

D

Pilus rod(rigid)

AdapterF

G

H

Tip fibrillum(flexible)

C Chaperone

A

fim operon

Found in human uropathogenic Escherichia coli (UPEC) strains

FimH adhesin has a basic specificity for mannose

FimH RecognizesTerminal Mannose Residues

A Serendipitous LigandReveals Remarkable Affinities

A Competition Assay for Affinity Measurements

Immobilized antibodytargetting the FimH

binding site

Carbohydrate ligands interfere with antibody

binding, preventingthe binding of FimH to

the chip

-7.62.3 µMmannose

-2.97.6 mMturanose

-2.612.8 mMsucrose

-6.131 µMfructose

-1.4100 mMgalactose

-2.89.24 mMglucose

-4.8300 µMmethyl 2-deoxy-α-D-mannopyranoside

∆G° (kcal/mol)KdLigand

Basic Affinity Pattern

Fructose, present for about 5% in fruit juices, inhibits type 1 fimbrial adherence

A Naturally Occurring Anti-Adhesive Compound

Inhibitor DesignAlkyl mannose

derivativesAromatic

substituents

Methylmannose(Man-C1)

Butylmannose(Man-C4)

Octyl-mannose(Man-C8)

Paranitrophenylmannose(PNP-Man)

Methylumbelliferylmannose(MU-Man)

-10.520MeUmbαMan

-10.044pNPαMan

-10.422octylαman

-11.35heptylαman

-10.910hexylαman

-10.425pentylαman

-9.3151butylαman

-8.9300propylαman

-8.11.2 103ethylαman

-7.72.2 103methylαman

-7.62.3 103mannose

∆G° SPR

(kcal/mol)

Kd SPR (nM)Ligand

A Linear Correlation Between Alkyl Chain Length and Energy

Energy

Num

ber

of

con

form

atio

ns

Docking Energy Histograms

Future Directions

Target tissue Glycoprotein mix

Mixture of fragments

Selection withlectin/adhesin

Binding fragments

Mass spectrometry anddatabase search

Identification

Treatment with proteases and glycosidases

Recovery and digestion

of glycoconjugates

Structure Thermodynamics

Experiments● BiaCore● ITC, DSC● Spectroscopic● titrations

A + B ABKa

G=-RT.ln(Ka)G=H-T.S

Ka=[AB]eq

[A]eq.[B]eqDocking

Structuredetermination● Crystallography● NMR

Integration

K88 Fimbriae

outer membraneD

Usher

G

Chaperone

?

E

Conclusions

OM

UsherChaperone

Conclusions

● X-ray crystallography reveals the structures of adhesins and their interactions with carobohydrate receptors in atomic detail

● Surface plasmon resonance measurements now enables the detection of the binding of mono- and oligosaccharides, using comparatively small amounts of protein and ligand

● Analysis of the combined data from these techniques provides insights in the specificity profiles of the adhesins, forming the basis for the design of novel adhesion inhibitors

Acknowledgements

Stefan OscarsonMartina LahmanRikard Slättegård

Ultrastructure Laboratory

Lode WynsRemy LorisJulie BouckaertHenri De GreveErwin De GenstJoris Messens

Stockholm/Goteborg Uppsala

Stefan KnightJenny BerglundAnton ZavialovDeva Choudhury

St. LouisChia HungScott Hultgren

Medimmune Solomon Langermann

Inge Van MolleAdinda Wellens

Fanny CoppensMike SleutelLieve CoolsPeter Bastaerts

Leiden Manfred Wuhrer André Deelder

Kourosch Abbaspour Therani

Organic Synthesis