Abstract
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).
Original language | English |
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Pages | A01-025 |
Publication status | Published - 2004 |
MoE publication type | Not Eligible |
Event | 5th International Conference on Biological Physics, ISBC 2004 - Gothenburg, Sweden Duration: 23 Aug 2004 → 27 Aug 2004 |
Conference
Conference | 5th International Conference on Biological Physics, ISBC 2004 |
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Country/Territory | Sweden |
City | Gothenburg |
Period | 23/08/04 → 27/08/04 |