Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure, backbone dynamics and protein association.

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155-270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of 1H-15N-correlated 2D HSQC, 15N-edited 3D TOCSY-HMQC, and 15N-edited 3D NOESY-HMQC spectra. By analysis of the alpha-proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain. To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.