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Fyn tyrosine kinase - 3-D structure and active site determination

One of the basic mechanisms for cell signaling is represented by tyrosine phosphorylation process. By this, metabolic pathways, cell growth and differentiation, membrane transport, apoptosis and other crucial processes within the cells are controlled. The enzymes involved in this are known as tyrosine kinases. Human Fyn tyrosine kinase (p59fyn) is one of the non-receptor tyrosine kinases belonging to Src-family kinases. It is activated after stimulation of T-cells by different triggers. Like in many other kinases, Fyn kinase catalytic domain is evolutionary very conserved. Therefore, it represents a good candidate for homology modelling and structure-based drug design (SBDD) techniques applications. There are some proteins belonging to the same class already present in Protein Data Bank (PDB) with well characterised 3-D structure. They can serve as a good starting models for further knowledge-based modelling. In this way accurate data on 3-D structure for not yet characterised proteins, can be obtained. In respect to Fyn, for this purpose, several template proteins have been used. They all have up to 70% sequence identity towards targeted protein - Fyn. COMPOSER suite of programs has been used in creating 3-D model. Quality of the obtained tertiary structure has been checked by two programs: PROCHECK and PROSAII. Knowledge about 3-D structure of Fyn and active site characterization, can help in using SBDD as a method of choice in designing its specific inhibitor. Staurosporine as a potent non-selective standard inhibitor of most tyrosine kinases has been used as ligand in Fyn active site characterization process. Its inhibitory activity on Fyn tyrosine kinase has been determinated in vitro by ELISA immunochemistry method. The obtained results were combined with results obtained in silico using predicted 3-D structure of Fyn. These data were compared with the literature avilable data on other kinase structures that have been (in most cases) experimentally obtained.

fyn; tyrosine kinase; homology modelling; COMPOSER; staurosporine

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