Molecular dynamics study of protein-oligosaccharide interaction mechanisms in chitinases engineered towards neolectins

Protein-carbohydrate interactions are essential in many biomolecular recognition events, such as inflammation, cell-cell recognition and adhesion, immunochemistry and human blood group type determination. A great deal of interest has thus arisen in isolation of medically important oligosaccharides. In this study our aim is to obtain atomic level information of the binding and to gain a deeper understanding of the factors determining the interactions between an oligosaccharide and a protein by using extended molecular dynamic (MD) simulations with explicit water. In addition to a conventional force field, a novel soft-core potential 1,2 originally developed for a priori modelling of surface loops of proteins without additional restraints was used here to the whole binding site of the modelled protein. The study concentrates on fungal 42kDa chitinase from Trichoderma harzianum 3, a naturally chitin degrading enzyme, containing an extended binding site providing a number of strong and specific interactions with up to 6-7 sugar units. Since these interactions come from a limited number of loops, the structure of chitinase (-barrel fold) provides an excellent platform for directed evolution studies. By mutating first the functional amino acid(s) and then altering the substrate specificity by locally directed saturation mutagenesis the functionality of a protein can be changed from a degrading enzyme to a specific binder. While experimentally determined three-dimensional structures were not available for the enzyme of interest, structural models were constructed based on the known structures of homologues. Experimentally determined sugar-protein complex structures of related chitinases were used in the initial simulations to evaluate the suitability of the force field parameters and simulation procedures. Classical MD (Gromacs) with a conventional force field and with a soft-core potential 1,2 is used to explore the conformational space of the chitinase loops and to study the functional behavior of the N-acetylglucosamine oligosaccharides and their derivates. Trajectories obtained from the simulations are used in analyzing the binding, especially the hydrogen bonding and hydrophobic interactions occurring via N/O-acetyl or O-methyl groups. The results from modeling are compared with the experimental data (mutagenesis, mass spectroscopy and nuclear magnetic resonance). Computational studies with the experimental work aim at development of neolectins, i.e. proteins selectively binding to given oligosaccharide structures, achieved by deactivating and engineering fungal chitinases towards the desired specificity and affinity. 1. Tappura, K.; Lahtela-Kakkonen, M; Teleman, O. J. Comput. Chem. 2000, 21(5), 388-97. 2. Tappura, K. Proteins 2001, 44(3), 167-79. 3. Boer, H.; Munck, N.; Natunen, J.; Wohlfahrt, G.; Söderlund, H.; Renkonen, O.; Koivula, A. (submitted 2004).