Crystal structure of human Fyn kinase domain complexed with non-specific inhibitor staurosporine

Department of Biological Science, Graduate School of Science, Osaka Prefecture University* Faculth of Bioenvironmental Science, Kyoto Gakuen University** Carna Bioscience***
â—‹Takayoshi Kinoshita* Mamoru Matsubara** Hiroshi Ishiguro*** Kouki Okita*** Toshiji Tada*

The tyrosine kinase Fyn is a member of the Src kinase family. Besides the role of Fyn in T cell signal transduction in concert with Lck, its excess activity in the brain is involved with conditions such as Alzheimer's and Parkinson's diseases. Therefore, inhibition of Fyn kinase may help counteract these nervous system disorders.
The truncated kinase domain (260-537) was overexpressed in the baculovirus expression system using Sf21 insect cells and purified using His Trap HP, Mono Q and Superdex 200 columns (GE Amersham Biosciences). The non-specific inhibitor staurosporine was suspended into the protein solution, and the complex solution was incubated at 277 K. Hexagonal-plate crystals of the complex were obtained at 277 K using a reservoir solution of 1.15 M ammonium phosphate dibasic, 0.2 M NaCl and 0.1 M imidazole buffer, pH 8.0. After dipping into Paratone-N oil (Hampton Research), the crystals were frozen using a nitrogen gas stream at 100 K. Diffraction data were collected at a wavelength of 1.0 Å using the synchrotron radiation at Photon Factory beamline NW12A. X-ray diffraction data were processed and scaled using the program HKL2000 (HKL). We solved the crystal structure of the complex at 2.8 Å resolution using the program AMoRe.
Staurosporine binds to the ATP-binding site of Fyn in a similar manner as in the Lck- and Csk- complexes. The structural basis for staurosporine binding to three protein tyrosine kinases, Fyn, Lck, and Csk may help in the development of new selective Fyn kinase inhibitors.