Engineering of PA-IIL lectin from Pseudomonas aeruginosa – Unravelling the role of the specificity loop for sugar preference
© Adam et al; licensee BioMed Central Ltd. 2007
Received: 13 February 2007
Accepted: 01 June 2007
Published: 01 June 2007
Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose – a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action.
In vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22–23–24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions – both of afore mentioned have strong effects on the saccharide preferences.
Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.
Carbohydrates are essential for life and in addition to their classical role as structural and energy storage components, they play crucial roles in many recognition events, signalling and communication processes. Recognition of glycoconjugates is an important event in biological systems and is frequently provided through protein-carbohydrate interactions. Many pathogens exploit host cell-surface glycoconjugates as receptors for attachment, tissue colonization and/or invasion. This is related to the fact that the structure of saccharides offers a wide range of diversity for encoding information, a fact directly tied to the variability of possible isomeric configurations of monosaccharides, increased even further when taking into account the possible linkages between the sugar units .
The recently heavily investigated area of protein-saccharide interactions holds a great potential of use in the field of drug design, namely targeting and delivery of active compounds to the proper site of action . Another therapeutical area involving lectin-saccharide interaction is the inhibition of bioadhesion, since bacteria are often able to effectively adhere to the surface membranes of the host cells via lectin binding, thus enabling subsequent colonization and advancement of the disease.
Pseudomonas aeruginosa is a gram negative bacterium and an opportunistic pathogen that infects immunocompromised patients and which is often involved in nosocomial infections . The bacterium is also responsible for chronic respiratory tract infections of individuals under mechanical ventilation and patients suffering from cystic fibrosis (CF). Carbohydrates play an important role in infections, since they are a target for bacterial binding through virulence factors such as adhesins, present on pili or flagella, and soluble lectins . In the particular case of CF patient lungs, an increase of fucosylation is observed, both at the levels of epithelia glycoconjugates  and altered mucins , and a fucose binding lectin from P. aeruginosa (PA-IIL) has been demonstrated to bind specifically to CF airline culture cells . The tetrameric lectin that was first identified and characterised from the P. aeruginosa cytoplasm  has been shown to be present in large quantities on the outer membrane of the bacteria and to be also involved in biofilm formation . Recently, it has been proposed that the lectin role lies in pilus genesis and proteolytic activity .
Through protein crystallography at high resolution, a precise three dimensional knowledge of the lectin has allowed the structural basis of the interaction between PA-IIL and fucose and fucose containing oligosaccharides to be understood [10, 11]. An unusual binding mode, mediated by two calcium ions, is responsible for the micromolar affinity for fucose which is much higher than that classically observed in protein-carbohydrate interactions . Previous inhibition tests have demonstrated that other monosaccharides are also recognised by PA-IIL with affinity in the decreasing order of L-fucose (Fuc) > L-galactose (L-Gal) > D-arabinose (Ara) > D-fructose (Fru) > D-mannose (Man) . Indeed, these monosaccharides all have either an L-galacto or a D-manno configuration that present two equatorial and one axial hydroxyl groups when in the 1C4 and 4C1 pyranose conformation, respectively. This particular stereochemistry is a requirement for the coordination of the two calcium ions as observed in the X-ray crystal structures of PA-IIL complexed with various monosaccharides [10, 12, 14, 15]. Precise equilibrium association constants, together with their enthalpic and entropic contributions, were evaluated for PA-IIL interacting with these monosaccharides and the crystal structures have been determined at very high resolution for the complexes with monosaccharides [10, 14].
This work presented here focuses on defining the fine specificity of PA-IIL and the influence of individual amino acids in the binding site for mannose/fucose which contribute to the lectin's preference. Considering the importance of the specificity loop, mutants of PA-IIL were designed. These mutants were constructed by replacing each of the amino acids of the so-called specificity loop, i.e. amino acids in positions 22, 23 and 24 for those present in RS-IIL (i.e, constructing mutants named S22A, S23A and G24N). Mutagenesis was followed by structural and thermodynamic studies to probe the importance of the specificity loop composition.
Expression and purification of recombinant proteins
Crystal structure of mutants and their complexes with monosaccharides
Data collection and refinement statistics
Resolution range (Å)
Highest resolution shell (Å)
a = 50.4 Å; b = 80.1 Å;
c = 52.5 Å; β = 109.6°
a= 52.6 Å; b = 74.9Å;
c = 53.5 Å; β = 114.7°
a = 52.5 Å; b = 73.0 Å;
c = 54.7 Å; β = 94.3°
a= 52.7 Å; b = 73.1Å;
c = 54.9 Å; β = 94.3°
a = 95.1 Å; b = 45.7 Å;
c = 88.0 Å; β = 94.2°
Matthews Coef (Å3/DA)
Solvent content (%)
R merge1,2 (%)
Wilson B-factor (Å2)
Rcrys3 (No. observations)
Rfree4 (No. observations)
Total number of atoms: Hetero/Protein/solvent
Number of side chains modelled with alternative conformations
Average refined B-factors per chains (A/B/C/D/water)
RMS deviation from ideality
Number of outliers on Ramachadran plot
PDB deposition code
Initial refinement of the crystal structures revealed that during soaking, the monosaccharides of interest had not always replaced the fucose molecules originally present in the protein complexes. Hence, amongst the four monomers that form the asymmetric unit, substitution of the sugar occurred in the four monomers of PA-IIL S23A/Me-α-Fuc and G24N/Me-α-Man, but only in three monomers of the S22A/Me-α-Man and S22A/Me-α-Fuc complexes and in only two monomers of G24N/Me-α-Fuc. The other two monomers of G24N/Me-α-Fuc are lacking both calcium ions and don't contain any sugar moiety in the binding site. Substitution of fucose by the mannoside was not successful at all during soaking crystals of PA-IILS23A/fucose in the Me-α-Man solution and the structure was not further refined.
Altogether five crystal structures have been refined, corresponding to the complexes of mutants S22A, S23A and G24N with Me-α-Fuc and S22A and G24N with Me-α-Man. Refinement statistics are summarised in Table 1. The sugar ring orientations of Me-α-Fuc and Me-α-Man in the binding site of mutants are similar to those observed in complexes with the wild type lectin [10, 15].
Structure of complexes of PA-IIL mutants and methyl α-L-fucopyranoside
In the binding site of all three mutants, the O2, O3 and O4 atoms of the methyl fucoside participate in the coordination spheres of the calcium ions, O3 occupying the space between the ions and interacting with both of them, whereas the O2 and O4 interact each with only one of the ions. The hydroxyl groups of the fucoside are involved in creating hydrogen bonds with the surrounding amino acids with O3 creating a bond to the side chain oxygen of Asp 99 and O4 to the carboxyl oxygen from Gly 114 belonging to the neighbouring monomer. These interactions are omnipresent in all the investigated mutants, as is the hydrophobic interaction between the methyl groups of the fucoside and of Thr 45.
Structure of complexes of PA-IIL mutants and methyl α-D-mannopyranoside
Comparison of contact distances (in Angstroms) in binding sites of the PA-IIL mutants
Man (O6) -- Ser23 (OG)
Man (O6) -- Asn24 (ND2)
Man (O6) -- Asp96 (OD2)
Man (O6) -- Ser23 (N)
Man (O6) -- Gly24 (N)
Asp96 (OD2) -- Ser22 (OG)
Asp96 (OD2) -- H2O (2)
H2O (2) -- Ser23 (OG)
Fuc (O1) -- H2O (1)
Thermodynamics of interactions of PA-IIL and its binding to monosaccharides
Thermodynamics of lectin/sugar binding. Thermodynamics of binding for different monosaccharides interacting with PA-IIL and its mutants, compared to thermodynamic characteristics of mannose-preferring lectin RS-IIL from Ralstonia solanacearum as determined by ITC at 25°C. Standard deviations calculated from three independent measurements for all ligands.
524 (± 36.7)
0.19 (± 0.03)
-38.4 (± 0.2)
-40.1 (± 0.7)
1.8 (± 0.7)
418 (± 72.8)
0.25 (± 0.01)
-37.8 (± 0.4)
-33.9 (± 1.2)
-3.8 (± 0.9)
235 (± 8.0)
0.43 (± 0.01)
-36.4 (± 0.1)
-41.3 (± 1.0)
4.9 (± 1.0)
26.4 (± 4.9)
3.96 (± 0.91)
-30.9 (± 0.5)
-37.1 (± 0.6)
6.2 (± 0.9)
23.8 (± 3.4)
4.3 (± 0.6)
-30.6 (± 0.4)
-26.9 (± 0.2)
-3.7 (± 0.6)
15.2 (± 2.3)
6.6 (± 0.9)
-30.0 (± 0.4)
-31.1 (± 0.4)
1.1 (± 0.7)
12.0 (± 1.0)
8.3 (± 0.7)
-29.0 (± 0.1)
-27.7 (± 0.5)
-1.3 (± 0.5)
10.3 (± 0.4)
9.7 (± 0.4)
-28.6 (± 0.1)
-26.8 (± 0.7)
-1.8 (± 0.6)
85.0 (± 5.0)
1.17 (± 0.07)
-33.8 (± 0.1)
-31.5 (± 0.6)
-2.3 (± 0.7)
82.2 (± 18.8)
1.29 (± 0.35)
-33.7 (± 0.6)
-18.7 (± 0.3)
-15.0 (± 0.9)
81.6 (± 14.4)
1.23 (± 0.07)
-33.9 (± 0.4)
-26.8 (± 1.7)
7.1 (± 1.3)
6.75 (± 0.31)
15.0 (± 0.66)
-27.6 (± 0.5)
-19.2 (± 0.2)
-8.3 (± 0.3)
438 (± 2.0)
0.23 (± 0.01)
-37.9 (± 0.1)
-24.9 (± 0.2)
-13.0 (± 0.2)
36.0 (± 1.8)
2.78 (± 0.14)
-31.7 (± 0.1)
-24.3 (± 0.6)
-7.4 (± 0.7)
2.34 (± 0.17)
42.9 (± 3.12)
-24.9 (± 0.2)
-27.1 (± 0.8)
2.1 (± 0.9)
1.91 (± 0.33)
54.0 (± 9.32)
-24.4 (± 0.4)
-16.7 (± 2.3)
-7.7 (± 2.7)
1.42 (± 0.05)
71.0 (± 3.00)
-23.7 (± 0.1)
-17.8 (± 1.3)
-5.9 (± 0.3)
As expected, the stoichiometry of binding was always close to four molecules of saccharide for one tetrameric unit of the lectin. For some protein batches, binding stoichiometry was around 3 signalling a possible deactivation of one subunit. Nevertheless, the other binding characteristics were not significantly affected by this and data evaluation clearly showed that all binding sites are equal and independent to each other.
The observed values of association constants for Me-α-Fuc varied as a function of the specificity loop mutation. The mutants S23A and G24N show approximately 200% and 160% higher affinity compared to PA-IIL, whose dissociation constant equals 430 nM. The S22A mutant displays a significant drop to a mere 11% of the PA-IIL affinity, its dissociation constant being consequently almost ten fold higher (3.96μM). Still, it is a considerably larger affinity than is displayed for the binding of Me-α-Fuc by the mannose-preferring lectin RS-IIL (the "non-detectable" value is lower than 1.10-4 M).
The results for non-methylated fucose have shown that the absence of methyl group at the anomeric position leads to an affinity decreased around one order of magnitude in the case of PA-IIL and mutants S23A and G24N. This effect is a general observation in protein-carbohydrate interactions. For S22A, the affinity drop was only slight, changing from 3.96 μM to 9.7 μM in terms of dissociation constant.
For Me-α-L-Gal, the affinity of the PA-IIL is approximately four times lower than in the case of Me-α-Fuc, with a dissociation constant of 1.17 μM . Comparing the affinity of the mutants towards Me-α-Gal, the mutants S23A and G24N displayed almost the same level of affinity as the wild type protein, with dissociation constants of 1.23 and 1.29 μM, respectively, whilst the mutant S22A has a considerably lower affinity of less than one tenth of it. The affinity towards methyl galactoside is generally lower than to methyl fucoside.
The situation is different when it comes to Me-α-Man. Overall affinity towards this monosaccharide is considerably lower than the previous ones. The values of dissociation constants are one hundred times higher than in the case of methyl fucoside, again comparable for the wild type and mutants S23A and G24N. On the other hand, the affinity towards mannoside increased approximately thirty times for the mutant S22A. Nevertheless, even for the S22A mutant, the affinity value is approximately ten times lower than for the mannose-preferring lectin RS-IIL.
In general, interactions between proteins and carbohydrates present a balance of hydrogen bonds and hydrophobic contacts. PA-IIL is a rather unique case since its interaction with fucose is established mainly through coordination by calcium ions and hydrogen bonds. The only hydrophobic interaction is established between the methyl group at C5 of fucose and the side chain of Thr45. The contribution of functional groups at the C5 position of fucose was already discussed in a previous article showing that hydrophobic interactions are not crucial for sugar binding . The relative contributions of the different thermodynamic parameters are more difficult to rationalise. The entropic term is a sum of multiple effects. A favourable entropic contribution is obtained when the increase of solvent entropy coming from water driven out of the site is correlated with minimal loss of conformational degrees of freedom in either ligand or protein chains during binding. Protein-carbohydrate interactions cannot usually meet these criteria because of the strong entropy penalty usually observed. Interestingly, the study of the sugar free PA-IIL structure has shown the presence of three water molecules in the binding site, strongly coordinated to the calcium ions . These water molecules are replaced by three hydroxyl groups in the complex with saccharide. The release of the water molecules, that are bound more strongly than in a classical carbohydrate binding site, is the reason of aforementioned behaviour and entropy terms closed to zero.
Complexes of mutants with methyl α-L-fucopyranoside
Previous work regarding the comparison of PA-IIL and RS-IIL has hinted that the reason behind the different affinities may be in the composition of the specificity binding loop. These observations are further confirmed by the results of the current experiments that point to a very special role for Ser22. In the absence of the side-chain hydroxyl group (mutant S22A), there is no hydrogen bond to Asp96 and the available carboxyl group of Asp96 binds a water molecule. This is in accordance with the observed entropy penalty.
Mutant S23A is even more interesting. The affinity towards methyl fucoside is almost two times higher than in the case of the native protein. Moreover, there is a significant difference in the contribution of both components of Gibbs energy. The native protein displays an unfavourable entropic contribution TΔS of -4.9 kJ/mol, whereas the mutant shows a favourable entropic part of 3.8kJ/mol, making the total difference 8.7kJ/mol. The hydroxyl group of Ser23 in the native protein interacts with the fucoside O5, creating an unfavourable entropy contribution. If this interaction was absent, the entropic contribution would become favourable for the free energy and consequentially, for the affinity. By replacing Ser23 with alanine, another interaction is also generated: the hydrophobic interaction between the side chain of alanine and the C5 methyl group of fucose. The sugar free structure of PA-IIL shows some flexibility of Ser23 which becomes specifically oriented during sugar binding and increases the contribution to the entropy penalty once sugar is bound. A comparison of thermodynamic data of all lectins from the PA-IIL family lacking serine at position 23, (S23A, RS-IIL and fucose-preferring lectin CV-IIL from Chromobacterium violaceum that was described recently ) shows a total positive entropy contribution to binding in all cases. This could imply that the presence of amino acids in the binding site, which don't lose their flexibility during binding of a sugar moiety and are not crucial for the interaction can favourable influence the entropy term. Findings relating to the residue at position 23 may be very useful in further mutagenesis and lectin-engineering experiments.
As for the mutant G24N, the situation is slightly different. There are no apparent new interactions observed and the two fold increase in affinity is therefore difficult to rationalize. While enthalpic contribution is almost the same, the entropy term becomes less unfavourable (difference of about 3 kJ/mol), which could imply that the mutant protein shows better surface complementarity to the interacting sugar. The side chain of Asp 24 makes an additional hydrogen bond with carboxylic group of Asp96 and closes the binding pocket that therefore cannot accommodate any further water molecule that is seen in all four binding sites of the wild type protein in proximity of Asp96.
Complexes of mutants with methyl α-D-mannopyranoside
The analysis of the crystal structure offers a possible explanation. The oxygen O6 of mannoside, forced to point out from the molecule by the presence of serine 22 (like in the case of the wild type protein), participates in a new hydrogen bond interaction with the amino group of the newly present Asn24. This results in a higher value of association constant. The amino group of Asn24 also makes a bridging interaction with Asp96 via the capture of one molecule of water. Although the crystal structure of the S23A mutant with mannoside is not available at the moment, the reasoning used in the cases of G24N and the wild-type lectin can be used also here for the explanation of difference in thermodynamic properties between fucoside- and mannoside-interaction.
Understanding and rationalising the structural and thermodynamic basis of interaction between lectins from pathogens and carbohydrates opens the door to designing high affinity inhibitors. On the other hand, detailed depiction of the interaction and description of crucial amino acids for affinity and/or specificity of the lectins can lead to targeted protein-engineering of the carbohydrate-binding proteins and fine tuning their properties for biotechnology, pharmacology and bioanalytical applications. Mutagenesis of amino acids forming the specificity binding loop of the high affinity lectin from P. aeruginosa allowed the amino acids responsible for sugar preference to be determined. This knowledge could initiate further studies using a combination of rational site directed mutagenesis and directed evolution with the goal of preparing lectins with advanced and novel properties.
Site-directed mutagenesis and expression
A construct named pET-25(b+)pa2l, containing the plasmid pET-25(b+) (Novagen) and the full-length wild type P. aeruginosa PA-IIL encoding gene , was used as a template. Site-directed mutagenesis was performed with the QuikChange™ Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer's instructions. The oligonucleotides used for the mutations were constructed as follows:
Mutant PA-IILS22A, primer pair PA-IILS22Asense/antisense (5'-CCG CCT TCG CCA ACG CGT CCG GAA CCC AGA CGG-3'/5-CCG TCT GGG TTC CGG ACG CGT TGG CGA AGG CGG-3'); mutant PA-IILS23A, primer pair PA-IILS23Asense/antisense (5'-CCG CCT TCG CCA ACT CGG CCG GAA CCC AGA CGG-3'/5'-CCG TCT GGG TTC CGG CCG AGT TGG CGA AGG CGG-3'); mutant PA-IILG24N, primer pair PA-IILG24Nsense/antisense (5'-CCG CCT TCG CCA ACT CGT CCA ACA CCC AGA CGG TGA ACG-3'/5'-CGT TCA CCG TCT GGG TGT TGG ACG AGT TGG CGA AGG-3'). Mutations were confirmed by DNA sequencing and new constructs were transformed into the E. coli Tuner(DE3) strain (Novagen).
Purification of recombinant wild-type and mutant PA-IIL proteins
Wild type PA-IIL lectin was expressed in E. coli BL21(DE3) (Novagen) and purified on a mannose-agarose column as previously described . PA-IIL mutants were prepared in similar way. E. coli Tuner (DE3) cells harbouring the plasmid encoding the desired mutant protein were grown at 37°C until OD600 = 0.6. After a three hour induction by 0.5 mM isopropylthiogalactoside at 30°C cells were harvested, disrupted by sonication and the supernatant applied to a D-mannose-agarose column (Sigma-Aldrich, USA) equilibrated by buffer composed of 20 mM Tris/HCl, 100 mM NaCl, 100 μM CaCl2, pH 7.5. After washing out unbound proteins, mutant proteins were eluted from the column using the same buffer containing 0.1 M D-mannose. Afterwards, the proteins were purified to homogeneity by the rapid, one step purification procedure previously described . The purified proteins were dialysed against distilled water for 1 week (for the removal of D-mannose) and stored at -20°C in lyophilized form.
Crystallization and structure determination of PA-IIL mutants
Mutants of PA-IIL were crystallised in complexes with fucose as their co-crystallisation with other sugars were not successful. The crystals were grown using the hanging drop vapour-diffusion method and conditions screened using the Hampton screens I and II. The best crystals obtained were soaked for 24 h in 10 mM Me-α-D-Man and Me-α-L-Fuc in the precipitant solution.
The different mutants gave co-crystals with fucose using the following conditions: 0.2 M ammonium sulphate, 30% PEG 4000 (G24N/Me-α-Fuc), 0.1 M HEPES (pH 7.5), 10 % isopropanol, 20% PEG 4000 (G24N/Me-α-Man), 0.2 M ammonium sulphate, 0.1 M Tris/HCl (pH 8.5), 30% PEG 4000 (S22A/Me-α-Man), 0.2 M lithium sulfate, 0.1 M Tris/HCl (pH 8.5), 30% PEG 4000 (S22A/Me-α-Fuc), 0.2 M calcium chloride, 0.1 M sodium acetate (pH 7.5), 20% isopropanol (S23A/Me-α-Fuc). Crystals, after soaking, were cryo-protected by soaking in 25 % glycerol v/v in the precipitant solution for as short a time as possible and were subsequently cryo-cooled to 100 K. All data images were recorded on beamlines ID14-2 or ID29 at the ESRF (Grenoble, France). The collected data were processed with MOSFLM  and scaled and converted to structure factors using the CCP4 program suite. The phases were determined by the molecular replacement technique with the MOLREP program of CCP4 using the monomeric PA-IIL/Fuc structure (PDB code 1UZV)  stripped of water molecules, calcium ions and sugar ligands as the search probe. All complexes contain tetramers in the asymmetric units. Initial structures were entirely rebuilt automatically using ARP/warp  and refinement was performed with REFMAC .
The mutations were verified using fo-fc and 2fo-fc electron density maps after molecular replacement using the native structure. Alternating cycles of refinement with manual construction using Coot  of missing residues, alternative conformations, water molecules, calcium ions and sugar ligands resulted in the final structures.
Coordinates and structure factors of the mutant complexes with Me-α-D-Man and Me-α-L-Fuc have been deposited in the Protein Data Bank under following codes: 2JDU (G24N/Me-α-Fuc), 2JDY (G24N/Me-α-Man), 2JDN (S22A/Me-α-Man), 2JDM (S22A/Me-α-Fuc) and 2JDP (S23A/Me-α-Fuc). Molecular drawings were prepared with PYMOL .
Titration calorimetry experiments were performed using a Microcal (Northampton, MA) VP-ITC microcalorimeter. All titrations were made in 0.1 M Tris-HCl buffer containing 30 μM CaCl2 (pH 7.5) at 25°C. Aliquots of 10 μL of each carbohydrate, dissolved in the same buffer, were added at 8 min intervals to the lectin solution present in the calorimeter cell. In the titrations, the protein concentration in the cell varied from 0.06 to 0.2 mM. The sugar concentration in the 250 μl syringe was 10–20 times higher than the protein concentration used in the experiment (from 1.2 to 3.5 mM) The temperature of the cell was kept at 25 ± 0.1°C. Aliquots of 15 and 10 μl of the ligand solution were added in 8 min intervals to the lectin solution present in the calorimeter cell. Data from a control experiment performed via identical injections of monosaccharide into the cell containing only a buffer were subtracted prior to data analysis. Integrated heat effects were analyzed by nonlinear regression using a single site binding model (Microcal Origin 7). Fitted data yielded the association constant (Ka) and the enthalpy of binding (ΔH). Other thermodynamic parameters, i.e., changes in free energy (ΔG) and entropy (ΔS), were calculated from the equationΔG = ΔH – TΔS = RT ln Ka
where T is the absolute temperature and R = 8.314 J mol-1 K-1. At least three independent titrations were performed for each ligand that was tested.
lectin II from Pseudomonas aeruginosa, RS-IIL, mannose-binding lectin from Ralstonia solanacearum
fucose-binding lectin from Chromobacterium violaceum
S23A and G24N, PA-IIL lectin mutated on positions 22, 23 and 24
We thank the ESRF, Grenoble for access to synchrotron data collection facilities and ICMG for financing microcalorimetry equipment. The work was supported by Ministry of Education (MSM0021622413) and Grant Agency of the Czech Republic (GA303/06/570 and doctoral grant GD204/03/H016), by CNRS and by French Association Vaincre la Mucoviscidose. Travels and visits between NCBR and CERMAV are supported by a BARRANDE exchange program.
- Gabius HJ, Siebert HC, Andre S, Jimenez-Barbero J, Rudiger H: Chemical biology of the sugar code. Chembiochem 2004, 5(6):740–764. 10.1002/cbic.200300753View ArticlePubMedGoogle Scholar
- Bies C, Lehr CM, Woodley JF: Lectin-mediated drug targeting: history and applications. Advanced drug delivery reviews 2004, 56(4):425–435. 10.1016/j.addr.2003.10.030View ArticlePubMedGoogle Scholar
- Gilboa-Garber N: Pseudomonas aeruginosa lectins. Methods Enzymol 1982, 83: 378–385.View ArticlePubMedGoogle Scholar
- Imberty A, Mitchell EP, Wimmerova M: Structural basis of high-affinity glycan recognition by bacterial and fungal lectins. Curr Opin Struct Biol 2005, 15(5):525–534. 10.1016/j.sbi.2005.08.003View ArticlePubMedGoogle Scholar
- Rhim AD, Stoykova LI, Trindade AJ, Glick MC, Scanlin TF: Altered terminal glycosylation and the pathophysiology of CF lung disease. J Cyst Fibros 2004, 3: 95–96. 10.1016/j.jcf.2004.05.021View ArticlePubMedGoogle Scholar
- Roussel P, Lamblin G: The glycosylation of airway mucins in cystic fibrosis and its relationship with lung infection by Pseudomonas aeruginosa. Adv Exp Med Biol 2003, 535: 17–32.View ArticlePubMedGoogle Scholar
- Stoykova LI, Ellway J, Rhim AD, Kim DJ, Glick MC, Scanlin TF: Binding of Pseudomonas aeruginosa lectin LecB to cystic fibrosis airway cells is inhibited by fucosylated compounds: Implications for therapy. Glycobiology 2005, 15(11):1228–1228.Google Scholar
- Tielker D, Hacker S, Loris R, Strathmann M, Wingender J, Wilhelm S, Rosenau F, Jaeger KE: Pseudomonas aeruginosa lectin LecB is located in the outer membrane and is involved in biofilm formation. Microbiology 2005, 151(Pt 5):1313–1323. 10.1099/mic.0.27701-0View ArticlePubMedGoogle Scholar
- Sonawane A, Jyot J, Ramphal R: Pseudomonas aeruginosa LecB is involved in pilus biogenesis and protease IV activity but not in adhesion to respiratory mucins. Infection and Immunity 2006, 74(12):7035–7039. 10.1128/IAI.00551-06PubMed CentralView ArticlePubMedGoogle Scholar
- Mitchell EP, Sabin S, Snajdrová L, Pokorná M, Perret S, Gautier C, Hofr C, Gilboa-Garber N, Koca J, Wimmerová M, Imberty A: High affinity fucose binding of Pseudomonas aeruginosa lectin PA-IIL: 1.0 A resolution crystal structure of the complex combined with thermodynamics and computational chemistry approaches. Proteins: Struct Funct Bioinfo 2005, 58(3):735–748. 10.1002/prot.20330View ArticleGoogle Scholar
- Perret S, Sabin C, Dumon C, Pokorna M, Gautier C, Galanina O, Ilia S, Bovin N, Nicaise M, Desmadril M, Gilboa-Garber N, Wimmerova M, Mitchell EP, Imberty A: Structural basis for the interaction between human milk oligosaccharides and the bacterial lectin PA-IIL of Pseudomonas aeruginosa. Biochem J 2005, 389(Pt 2):325–332.PubMed CentralView ArticlePubMedGoogle Scholar
- Mitchell E, Houles C, Sudakevitz D, Wimmerova M, Gautier C, Pérez S, Wu AM, Gilboa-Garber N, Imberty A: Structural basis for oligosaccharide-mediated adhesion of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. Nature Struct Biol 2002, 9: 918–921. 10.1038/nsb865View ArticlePubMedGoogle Scholar
- Garber N, Guempel U, Gilboa-Garber N, Doyle RJ: Specificity of the fucose-binding lectin of Pseudomonas aeruginosa. FEMS Microbiol Lett 1987, 48(3):331–334. 10.1111/j.1574-6968.1987.tb02619.xView ArticleGoogle Scholar
- Sabin C, Mitchell EP, Pokorna M, Gautier C, Utille JP, Wimmerova M, Imberty A: Binding of different monosaccharides by lectin PA-IIL from Pseudomonas aeruginosa: Thermodynamics data correlated with X-ray structures. FEBS Lett 2006, 580(3):982–987. 10.1016/j.febslet.2006.01.030View ArticlePubMedGoogle Scholar
- Loris R, Tielker D, Jaeger KE, Wyns L: Structural basis of carbohydrate recognition by the lectin LecB from Pseudomonas aeruginosa. J Mol Biol 2003, 331(4):861–870. 10.1016/S0022-2836(03)00754-XView ArticlePubMedGoogle Scholar
- Sudakevitz D, Kostlanova N, Blatman-Jan G, Mitchell EP, Lerrer B, Wimmerova M, Katcof DJ, Imberty A, Gilboa-Garber N: A new Ralstonia solanacearum high affinity mannose-binding lectin RS-IIL structurally resembling the Pseudomonas aeruginosa fucose-specific lectin PA-IIL. Mol Microbiol 2004, 52(3):691–700. 10.1111/j.1365-2958.2004.04020.xView ArticlePubMedGoogle Scholar
- Wiseman T, Williston S, Brandts JF, Lin LN: Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 1989, 179(1):131–137. 10.1016/0003-2697(89)90213-3View ArticlePubMedGoogle Scholar
- Pokorna M, Cioci G, Perret S, Rebuffet E, Kostlanova N, Adam J, Gilboa-Garber N, Mitchell EP, Imberty A, Wimmerova M: Unusual entropy-driven affinity of Chromobacterium violaceum lectin CV-IIL toward fucose and mannose. Biochemistry 2006, 45(24):7501–7510. 10.1021/bi060214eView ArticlePubMedGoogle Scholar
- Leslie AGW: Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography 1992., 26: [http://www.mrc-lmb.cam.ac.uk/harry/mosflm/]Google Scholar
- COLLABORATIVE COMPUTATIONAL PROJECT:The CCP4 Suite: Programs for protein crystallography Acta Crystallogr D Biol Crystallogr 1994, 50(Pt 5):760–763.
- Perrakis A, Morris R, Lamzin VS: Automated protein model building combined with iterative structure refinement. Nat Struct Biol 1999, 6(5):458–463. 10.1038/8263View ArticlePubMedGoogle Scholar
- Murshudov GN, Vagin AA, Dodson EJ: Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 1997, 53(Pt 3):240–55. 10.1107/S0907444996012255View ArticlePubMedGoogle Scholar
- Emsley P, Cowtan K: Coot: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004, 60(Pt 12):2126–2132. 10.1107/S0907444904019158View ArticlePubMedGoogle Scholar
- DeLano WL: The PyMOL molecular graphics system.DeLano Scientific, San Carlos, CA; 2003. [http://www.pymol.org]Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.