THERMODYNAMICAL ANALYSIS OF THE MUTANT CV-IIL LECTINS FROM CHROMOBACTERIUM VIOLACEUM
Eva Dejmková 1, Martina Pokorná 1, Michaela Wimmerová1,2
Centre for Biomolecular Research and 2Department of Biochemistry,
Lectins are proteins or glycoproteins of non-immune origin capable agglutinate cells. They are able to specifically recognize saccharides with high affinity. However, the major function of lectins appears to be in the cell recognition process, they generally recognize diverse sugar structures and mediate a variety of biological processes. Bacterial lectins play a crucial role in recognition of sugar moieties on the host cell surface and consequently can cause bacterial adhesion. An attractive approach is the use of agents that interfere with the ability of the bacteria to adhere to the host cell surface, since such adhesion is one of the initial stages of the infectious process. However, the process of ligand binding is very complex and complicated, deeper understanding of the thermodynamics of lectin-saccharide interaction can be useful for the molecular design of potent anti-adhesion therapeutics.
Chromobacterium violaceum is an opportunistic pathogen that commonly occurs in water and soil in tropical and subtropical regions. Infection caused by this bacterium can be fatal for immunocompromised people and children. Bacterial unusual resistance to antibiotics is the reason of high mortality.
The lectin from C. violaceum named CV-IIL is a tetramer (the structural functional unit is a dimer), each monomer is composed of 113 amino acids. This lectin shows ability to bind L-fucose (6-deoxy-L-galactose) and D-mannose with high affinity. Each monomer contains two calcium ions in the carbohydrate binding site, which mediate binding of the saccharides. The crystal structure of CV-IIL demonstrates that there is also one water molecule, which plays a special role by bridging sugar with backbone nitrogen atom and also with side chain of the amino acid threonine in position 97. (Fig.1)  Therefore, several representatives from different groups of amino acids were chosen for the mutagenesis in this position 97.
The complete thermodynamical analysis was
performed using isothermal titration microcalorimetry method (ITC 200,
Microcal). From the single measurement we can obtain and consequently calculate
important thermodynamical values (enthalpy, entropy, Gibbs free energy) as well
as binding affinity and stoichiometry of the lectin-saccharide interaction. The
interaction with high affinity ligand α-Me-fucoside was measured in case
of each prepared mutant lectin at different temperatures in the range from 10
∆S° = ∆S solv + ∆S config + ∆S rot + ∆S transl 
The two major contributions to the binding entropy are the change in conformational and the solvation entropy, which will be discussed. However, the values of the Gibbs free energy are very similar, the difference in enthalpy and entropy contributions were marked in some cases.
The work is supported by Ministry of
Education (MSM0021622413, ME08008) and Czech Science Foundation (GA/303/09/1168)
1. Pokorná M., Cioci G., Perret S, Rebuffet E., Kostlánová N., Adam J., Gilboa-Garber N., Mitchell E.P., Imberty A., Wimmerová M., Unusual Entropy-Driven Affinity of Chromobacterium violaceum Lectin CV-IIL toward Fucose and Mannose, Biochemistry 45 (24) 7501 - 7510, 2006
2. Chervenak M.C., Toone E.J., Calorimetric Analysis of the Binding of Lectin with Overlapping Carbohydrate-Binding Ligand Specifities, Biochemistry 34 5685 - 5695, 1995