title: The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic NMR spectroscopy.
abstract: The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signaling processes from focal adhesions. The phosphorylated tyrosine residue 397 in FAK is able to bind the Src SH2 domain. To establish the extent of the FAK binding motif, the binding affinity of the SH2 domain for phosphorylated and unphosphorylated FAK-derived peptides of increasing length was determined and compared with that of the internal Src SH2 binding site. It is shown that the FAK peptides have higher affinity than the internal binding site and that seven negative residues adjacent to the core SH2 binding motif increase the binding constant 30-fold. A rigid spin-label incorporated in the FAK peptides was used to establish on the basis of paramagnetic relaxation enhancement whether the peptide-protein complex is well defined. A large spread of the paramagnetic effects on the surface of the SH2 domain suggests that the peptide-protein complex exhibits dynamics, despite the high affinity of the peptide. The strong electrostatic interaction between the positive side of the SH2 domain and the negative peptide results in a high affinity but may also favor a dynamic interaction.
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
authors.forename: Hanna E
authors.initials: HE
authors.lastname: Lindfors
authors.forename: Jan Wouter
authors.initials: JW
authors.lastname: Drijfhout
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry; D011485:Protein Binding; D013499:Surface Properties; D013816:Thermodynamics; D017186:Titrimetry; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry*
chemical_list: D010748:Phosphopeptides; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases