J. Alán, P. Kulhánek and J. Koča


National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic


Binding free energy is the important thermodynamic property used for description of affinity of ligand to receptor. It is used in drug design for fast qualitative ranking of drug-like compounds. Experimental laboratory procedure leading to acquirement of binding free energy is multi step approach, which is long and possibly without results. Problems with purification and obtaining of efficient concentration of sample for measurement of thermodynamic parameters are most common. Thus free energy calculations are convenient supply laboratory measurements. When 3D structure of ligand receptor complex is available, then binding free energies can be calculated. Moreover affinities of structurally similar ligands can be then easily calculated. Most demanding objective in this field is preparation of universal procedure for calculations of carbohydrate affinities to lectins.

Potential of free energy calculations will be shown on project from glycobiochemistry field, particular on lectins from bacteria Pseudomonas aeruginosa and Chromobacterium violaceum. Lectins are group of proteins of non-immune origin specifically binding carbohydrates with high affinity. They play crucial role in cell recognition, signaling and adhesion. Bacterial lectins are known as very important participants in process of bacterial infiltration of host organism.

Lectin PA-IIL is a tetrameric protein where dimer is functional unit with two calcium ions in each binding site. This lectin mediates adhesion of bacteria Pseudomonas aeruginosa to tissues of respiratory tract of patients suffering from cystic fibrosis. PA-IIL shows highest affinity to L-fucose and binds also other carbohydrates [1]. Chromobacterium violaceum lectin CV-IIL is member of PA-IIL lectin superfamily and shows similar function. In silico designed and prepared mutant structures of this lectin were used for molecular dynamics simulations. Their affinity to α-L-fucose and α-D-mannose was evaluated to elucidate binding contributions of residues within binding pocket.  

The binding free energies of complexes of PA-IIL with α-L-fucose, Me-α-L-fucose and Me-α-L-galactose and complexes of CV-IIL and CV-IIL mutants with α-L-fucose α-D-mannose were estimated by thermodynamic integration method. This method is available in Amber 9 package [3]. It is based on slow alchemical transformation of one molecule to another. This method allows using explicit solvent model and parallelization of computations. Computed binding free energies of PA-IIL, CV-IIL and various monosaccharides were compared with isothermal titration microcalorimetry data. Computed relative binding free energies were used for compilation of series of ligand affinity to receptor which was in agreement with experimentally obtained data. Affinities computed by thermodynamic integration differ from experimental values in range of 0.1 to 1.0 kcal/mol.


This work has been supported by Ministry of Education of the Czech Republic (MSM0021622413, LC06030). The access to the METACentrum supercomputing facilities provided under the research intent MSM6383917201 is highly appreciated. The research leading to these results has received funding from the European Community's Seventh Framework Programme under grant agreement no. 205872. This work has been supported by Czech Science Foundation (301/09/H004, doctoral program).  


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[2] M. Pokorná, G. Cioci, S. Perret, E. Rebuffet, N. Kostlánová, J. Adam, N. Gilboa-Garber, E. P. Mitchell, A. Imberty & M. Wimmerová, Biochemistry, 45 (2006) 7501-7510, Unusual entropy-driven affinity of Chromobacterium violaceum lectin CV-IIL towards fucose and mannose.


[3] D. A. Case, T. A. Darden, T. E. Cheatham, C. L. Simmerling, J. Wang, R. E. Duke, R. Luo, K. M. Merz, D. A. Pearlman, M. Crowley, R. C. Walker, W. Zhang, B. Wang, S. Hayik, A. Roitberg, G. Seabra, K. F. Wong, F. Paesani, X. Wu, S. Brozell, V. Tsui, H. Gohlke, L. Yang, C. Tan, J. Mongan, V. Hornak, G. Cui, P. Beroza, D. H. Mathews, C. Schafmeister, W. S. Ross, P. A. Kollman, University of California, San Francisco, 2006, Amber 9.