This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 9483–9485 9483

Cite this: Chem. Commun., 2011, 47, 9483–9485

AFM investigation of Pseudomonas aeruginosa lectin LecA (PA-IL)

filaments induced by multivalent glycoclustersw

Delphine Sicard,aSamy Cecioni,

bcMaksym Iazykov,

aYann Chevolot,

aSusan E. Matthews,

d

Jean-Pierre Praly,bEliane Souteyrand,

aAnne Imberty,

cSebastien Vidal*

band

Magali Phaner-Goutorbe*a

Received 26th May 2011, Accepted 13th July 2011

DOI: 10.1039/c1cc13097h

Atomic force microscopy reveals that Pseudomonas aeruginosa

LecA (PA-IL) and a tetra-galactosylated 1,3-alternate

calix[4]arene-based glycocluster self-assemble according to an

aggregative chelate binding mode to create monodimensional

filaments. Lectin oligomers are identified along the filaments and

defects in chelate binding generate branches and bifurcations. A

molecular model with alternate 908 orientation of LecA tetramers

is proposed to describe the organisation of lectins and glycoclusters

in the filaments.

The opportunistic bacterium Pseudomonas aeruginosa is a

major cause of lung infections for immuno-compromised

and cystic fibrosis patients.1 Since the appearance of several

multidrug resistant strains, new therapeutic approaches are

receiving much attention especially for the prevention of

pathogen adhesion to host epithelia surfaces.2 In their

infection strategy, microorganisms often use carbohydrate-

binding proteins called lectins, to recognize and bind to host

cells.3 Pseudomonas aeruginosa displays two soluble lectins

(LecA/PA-IL and LecB/PA-IIL) which are implicated in

binding and virulence events leading to lung infection.4 A

structural study of LecA showed a homotetrametric lectin with

specificity for galactosides with a calcium ion involved in the

binding site.5 The molecular size of the tetramer can be described

as parallel piped with 7.0 � 3.2 � 1.9 nm3 dimensions (Fig. 1a).

The design of high affinity ligands for bacterial lectins6

such as LecA represents a strategy for the development

of anti-bacterial drugs, able to block the infection process

at the early stage of binding to the host cell.7 The affinity

of LecA for galactose is in the submillimolar range and the

design of multivalent glycoconjugates takes advantage of the

so-called ‘‘glycoside cluster effect’’,8 through the concept of

multivalency.9 We have recently synthesized topologically

isomeric calix[4]arene glycoclusters and measured nanomolar

affinities by microcalorimetry (ITC) and surface plasmon

resonance (SPR) for LecA.7c The sugar–protein interaction

was shown to be strongly dependent on both the valency and

the topology of the glycoclusters. The 1,3-alternate

calix[4]arene-based glycocluster (Fig. 1b) displayed the best

affinity towards LecA and molecular modeling was performed

in order to rationalize the high affinity observed and indicated

an aggregative chelate binding mode (Fig. 1c).

In order to further characterize the interaction between

calix[4]arene-based glycoclusters and LecA, we performed an

atomic force microscope (AFM) study of the self-assembly of

these partners. AFM offers the opportunity to evaluate both the

size and shape of nano-assemblies formed between multimeric

lectins and multivalent ligands. However, only a few experiments

have been reported where lectins and glycoconjugates were

deposited on a surface and then studied in air by AFM to

obtain additional information concerning their self-assembly

properties.10 In these recent reports, the sugar-lectin arrangement

yielded thin and thick films on the surface, which could then

self-assembly into a network rather than discrete assemblies.

Fig. 1 (a) Three-dimensional structure of lectin LecA (pdb code:

1OKO). Blue spheres represent calcium ions in binding sites. Dimensions

are measured between calcium ions. (b) Structure of the galactosylated

1,3-alternate calix[4]arene-based glycocluster 1. (c) Molecular modeling

pictures of aggregative chelate arrangement between LecA and the

ligand.7c Dimensions of the glycocluster 1 were measured between the

O-4 oxygen atom of galactose residues.

aUniversite de Lyon, Institut des Nanotechnologies de Lyon (INL), UMRCNRS 5270, site Ecole Centrale de Lyon, 36, avenue Guy de Collongue,69134 Ecully cedex, France. E-mail: magali.phaner@ec-lyon.fr

b Institut de Chimie et Biochimie Moleculaires et Supramoleculaires(ICBMS, UMR 5246), Laboratoire de Chimie Organique 2 –Glycochimie, CNRS, Universite de Lyon, 43 Boulevard du 11Novembre 1918, 69622 Villeurbanne, France.E-mail: sebastien.vidal@univ-lyon1.fr

cCERMAV – CNRS, UPR5301, affiliated with Universite Joseph Fourierand ICMG, BP 53, 38041, Grenoble, France

dUniversity of East Anglia, School of Pharmacy, Norwich, NR4 7TJ, UKw Electronic supplementary information (ESI) available. See DOI:10.1039/c1cc13097h

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9484 Chem. Commun., 2011, 47, 9483–9485 This journal is c The Royal Society of Chemistry 2011

Here we report the formation of supramolecular structures

through self-assembly of proteins mediated by small molecules.

In a typical AFM experiment, solutions containing 20 mL of

CaCl2 (0.3 mM in final concentration) and 10 mL of recombi-

nant LecA5b (25 pM final concentration) and 10 mL of tetra-

galactosylated glycocluster 1 (25 pM final concentration) were

mixed and incubated during 1 h, at room temperature, so that

the lectin and glycocluster could bind and equilibrate in

solution. The presence of calcium cation is required for an

active lectin binding site.5a The solution was then deposited on

a freshly cleaved mica surface and the sample dried overnight

in a desiccator, at ambient pressure, with silica gel as a desiccant.

Topography images were taken in air, at room temperature,

using a Di-Cp-II (Bruker) AFM microscope in the Amplitude

Modulation (AM) AFM mode with MikroMasch NSC 21 tips

(see ESIw). The data analysis was performed with Gwyddion

Software.

The AFM image of the complex between LecA and the

galactosylated glycocluster 1 displayed small filaments on the

surface (Fig. 2a). Negative controls performed for LecA incubated

with the corresponding tetra-mannosylated glycocluster7c did

not reveal the presence of any filaments (see ESIw). For LecA/

1 complexes, the images showed linear segments interrupted

by bifurcations and branching points. The length of the linear

segments varies between 90 and 500 nm � 5 nm. Branching

occurs mostly in linear regions with only one defect at a time

on a filament. The average height of these structures is estimated

at 1.7 � 0.5 nm which is in agreement with the thickness of the

dried protein (i.e. slightly less than 1.9 nm, vide supra) (Fig. 2b).

The average width of these monodimensional filaments is of

36.4 � 8.5 nm, which is 5- to 10-times more than the length

and width of the lectin as measured by X-ray crystallography.

This difference is believed to be a consequence of both the

curvature radius of the tip, which is known to enlarge features

in AFM, and the experimental adsorption conditions. In fact,

the lateral dimensions measured in AFM are obtained by a

convolution of the tip shape with the real size of the features.11

In addition, it is well known that biological objects adsorbed

on a substrate are spread over the surface to promote a better

binding.12 Our drying (one night in a desiccator) and imaging

conditions (in air) could also increase this spreading-out of the

biomolecules. The mica structure is hexagonal, its influence

would induce preferentially angles of 601, 1201. . .. However, a

statistical analysis of the angle distribution was performed on

all the images and no preferential angle was identified (distribu-

tion on 130 angles). This indicates that no influence of the mica

structure was observed confirming that filaments were formed

in the solution before deposition and evaporation.

We have previously shown7c that in terms of topology the

best molecular model of the interaction between LecA and the

1,3-alternate glycocluster 1 would be the aggregative chelate

binding mode (Fig. 1c). Two 1,3-alternate galactose epitopes

of the glycocluster 1 can chelate the two adjacent binding sites

of a lectin tetramer on the width side (3.2 nm). The two other

monosaccharides can have the same interaction with another

lectin tetramer. The repetition of this particular structure, by

self-assembly, which would lead to the formation of a mono-

dimensional filament is consistent with our AFM images

(Fig. 2a). The length of linear segments is in agreement with

a repeat of 10 to 50 LecA tetramers, based on the dimensions

of the modelled filament (Fig. 3c and ESIw).The branches observed between filaments can be rationalised

by a defect in the symmetry of the glycocluster. One of the four

galactose residues can then bind to a third lectin tetramer on

the side of the filament, generating a branching point (Fig. 2c).

A model was generated using the Sybyl software (Tripos, St.

Louis) by exploring the available conformational flexibility,

the length of the triethyleneglycol linker, and ensuring the

absence of steric conflict between adjacent proteins (see ESIw).The model demonstrated that such a branching point to

generate the formation of another filament binding to the first

one is possible, even though no predominant angle could be

clearly identified.

A higher resolution image showed the discrete LecA tetramers

along the linear filament (Fig. 3a) as rectangular patterns

positioned one next to each other. Profile measurements indicated

that a segment of 102 nm contains six of these patterns with

Fig. 2 (a) AM-AFM image of the filaments of lectin LecA and

glycocluster 1 on the mica substrate. Image size is of 850 � 850 nm2.

(b) Height of the filament on the profile (black bar in (a)). (c) Molecular

modeling of 12 lectin tetramers (cyan) connected by galactosylated

glycoclusters (dark blue). 8 tetramers have been modeled in lines, and

4 from a branching point.

Fig. 3 (a) High resolution AM-AFM image of the LecA/1 filament at

a 400 nm scan range on the mica surface. (b) Profile of six portions on

a filament (black line) and on a mica substrate (cyan line). Height of

the filament is not calibrated with the mica surface. (c) Details of the

molecular model of LecA tetramers linked by glycocluster 1 with the

corresponding schematic representation.

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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 9483–9485 9485

an average size of around 17 � 3.3 nm per pattern (Fig. 3b).

This value is 2 times greater than the theoretical length (9 nm,

longitudinal dimensions of LecA and glycocluster 1). Again,

the lateral size of each repeated tetramer cannot be precisely

estimated because of the limitations of the AFM method.

However, the enlargement of the pattern is less along the

filament than perpendicularly (36.4 � 8.5 nm) and cannot be

just attributed to a different geometry of the tip in both

directions but mostly to the spreading-out of the molecules

limited by the proximity of the neighboring molecules along

the filament.12 These values would suggest that the lectins are

arranged perpendicularly to the growth of the filament, this is

in contrast to the result obtained from models in which bridging

occurs on the shorter face of the lectin. This discrepancy is a

consequence of the imaging technique. The calculated arrange-

ment is believed to be the most probable, the binding sites are

too far away on the long side to allow perpendicular growth

(Fig. 3c). A difference in contrast was observed between the

patterns, some of them are brighter than the others, which

indicates a variation in the height value. A statistical measure-

ment on different patterns revealed an average height shift of

1.7 � 0.5 nm. This is consistent with our current model of the

LecA/1 complex7c which proposes that the cobblestone-shaped

tetramers are maintained in a 901 orientation one to the other,

due to the geometry of the calixarene-based glycocluster 1 in

which the galactose pairs on the top and bottom sides of the

calix[4]arene are in perpendicular planes (Fig. 1c). This topology

would result in a difference in height between neighbouring

tetramers as seen with the height shift of the AFM patterns.

However, the alternance of bright and dark patterns was not

systematic on the AFM image and one should also consider the

roughness of the mica surface underneath which could generate a

small variation of contrast (Fig. 3b).

In conclusion, the calixarene-based glycocluster previously

studied through bioanalytical techniques (HIA, ELLA, ITC

and SPR) for its binding properties towards the LecA was

further investigated by means of AFM to gain a more complete

understanding of its specific mode of binding. This AFM study

revealed that the aggregative chelate binding mode was most

probably adopted in the self-assembly of the glycocluster with

the lectin. A network of monodimensional filaments was

formed, with branching points and rare defects, attributed to

conformational changes in the glycosylated ligand. High

resolution images revealed each discrete lectin tetrameric unit

along the filament. Generally, the interaction between multimeric

lectins and multivalent ligands yields to the formation of aggre-

gates, or eventually organized 2D-networks.10a,13 Filament-like

association has been observed previously only for dimeric

bacterial BclA lectin interacting with bivalent mannosides.10b

The regular self-assembly through building blocks, observed

here, points to applications in nanotechnology. Further AFM

investigations are ongoing in our laboratories to describe more

precisely these self-assembly processes with various concentrations

of bio-materials, various lectin/glycocluster ratios, both in air

and in solution.

This work was financially supported by the CNRS, the

French Research Ministry with the ANR-08-BLAN-0114-01

programme, the LyonBioPole consortium and the French

Association ‘‘Vaincre laMucoviscidose’’ (against Cystic Fibrosis).

We also acknowledge University Claude Bernard Lyon 1, the

CNRS and University of East Anglia. S.C. thanks the Region

Rhone-Alpes for additional funding (Cluster de Recherche

Chimie).

Notes and references

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