Search

Background: 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. The study concentrates on fungal 42kDa chitinase from Trichoderma harzianum [1], 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. Methods: 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 [2,3] 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. Results and conclusions: 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 [2,3] 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] H. Boer, N. Munck, J. Natunen, G. Wohlfahrt, H. Söderlund, O. Renkonen, A. Koivula, submitted 2004. [2] K. Tappura, M. Lahtela-Kakkonen, O. Teleman, J. Comput. Chem. 21, 388 (2000). [3] K. Tappura, Proteins, 44, 167 (2001).

The binding affinity and specificity of recombinant antibodies can be modified by site-directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine-tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen-binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.

The cohesin-dockerin interaction in Clostridium thermocellum cellulosome mediates the tight binding of cellulolytic enzymes to the cellulosome-integrating protein CipA. Here, this interaction was used to study the effect of different cellulose-binding domains (CBDs) on the enzymatic activity of C. thermocellum endoglucanase CelD (1,4-β- d endoglucanase, EC 3.2.1.4 ) toward various cellulosic substrates. The seventh cohesin domain of CipA was fused to CBDs originating from the Trichoderma reesei cellobiohydrolases I and II (CBD CBH1 and CBD CBH2 ) (1,4-β- d glucan-cellobiohydrolase, EC 3.2.1.91 ), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBD Cex ) (β-1,4- d glucanase, EC 3.2.1.8 ), and from C. thermocellum CipA (CBD CipA ). The CBD-cohesin hybrids interacted with the dockerin domain of CelD, leading to the formation of CelD-CBD complexes. Each of the CBDs increased the fraction of cellulose accessible to hydrolysis by CelD in the order CBD CBH1 < CBD CBH2 ≈ CBD Cex < CBD CipA . In all cases, the extent of hydrolysis was limited by the disappearance of sites accessible to CelD. Addition of a batch of fresh cellulose after completion of the reaction resulted in a new burst of activity, proving the reversible binding of the intact complexes despite the apparent binding irreversibility of some CBDs. Furthermore, burst of activity also was observed upon adding new batches of CelD–CBD complexes that contained a CBD differing from the first one. This complementation between different CBDs suggests that the sites made available for hydrolysis by each of the CBDs are at least partially nonoverlapping. The only exception was CBD CipA , whose sites appeared to overlap all of the other sites.