ty of LecB this repulsive interaction is additionally avoided by a conformational change of the protein and Asp96 flips away from the b-substituent after 4 ns simulation time. This change is accompanied by the breakage of the hydrogen-bond network in the protein as seen in the time series of the distance between Asp96 and Ser22. This new position is, however, not optimal and additional transitions between the conformation with and without the hydrogen bond, called closed and open form in the following, occur during the simulations. Representative structures of the closed and open form are shown in 17 / 22 Molecular Basis of Monosaccharide Selectivity of LecB possible in the complex of 4 with LecB, however, with a perhaps different probability as for the complex of a-D-mannose with LecB. This phenomenon could originate from the additional methyl group in 4, which stabilizes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19691102/ the complex by van der Waals interaction with the protein. The importance of the van der Waals interaction of fucose with Thr45 in LecB has previously been analyzed by Mishra et al. In the same study, a similar flexibility of loops in the carbohydrate recognition domain, as observed here for the low TAK-438 (free base) affinity ligand, was reported in absence of the high affinity ligand fucose. Although the conformation of LecB is generally conserved in the lowtemperature crystal structures deposited in the protein data bank, flexibility at ambient conditions seems thus reasonable 
supporting our observations. To further validate these results, we are currently working on an experimental proof for the flexibility and rearrangement of the protein conformation. Conclusion We have dissected the contribution of individual substituents of the natural ligands fucosides and mannosides to binding with LecB. The lipophilic interaction of the methyl group of fucose and derivatives increases binding affinity by a factor of 46 compared to analogs lacking this methyl group. A combination substituents presumably forming attractive interactions from 1 and 2 with LecB into hybrid 4, i.e., PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19692133 the equatorial methyl group of fucose and the hydroxymethyl group of mannose, improved binding affinity with respect to mannosides, but surprisingly did not lead to a synergistic effect in binding affinity. Although the hydroxymethyl group in 2 forms an additional hydrogen bond with the lectin in the crystalline low temperature state, our thermodynamic data for 4 and its deoxygenated analog 6 suggest only a minor contribution to binding in aqueous solution. In addition, we conclude from our experimental data and theoretical calculations, that the steric demand of equatorial substituents at the superimposing positions of fucose-C1 and mannose-C5 leads to unfavorable steric interactions with Asp96 and to a high destabilization of the protein surroundings, and in this way, accounts for the reduced affinity of mannosides and derivatives with respect to fucosides. 18 / 22 Molecular Basis of Monosaccharide Selectivity of LecB In the LecB orthologs RS-IIL and BclA, a mutation in the so-called sugar specificity loop of Ser22 in LecB to alanine results in an electrostatic void within the protein, which is filled by mannose O-6 and establishing of the hydrogen bond with Asp96. This explains the higher selectivity of these orthologs for mannose over fucose in contrast to LecB. In case of LecB, the additional hydrogen bond of a-D-mannose with Ser23 observed in the crystal structure, and the entropic gain due to the higher flexibili
Posted inUncategorized