title: A Focal Adhesion Kinase-Derived Peptide Binds the Src SH3 Domain in Two Orientations, As Demonstrated Using Paramagnetic Nuclear Magnetic Resonance.
abstract: SH3 binding peptides contain polyproline helices and are classified according to their binding orientations as N-to-C-terminal or C-to-N-terminal. We have tested the hypothesis that such a peptide binds in both orientations but with different populations. A focal adhesion kinase (FAK)-derived peptide was tested for its binding orientation on the Src SH3 domain. Paramagnetic tags were introduced at several positions on the SH3 domain, and on the basis of the paramagnetic relaxation enhancements (PREs) of the amide protons, the positions of the paramagnetic centers were determined. Two peptides were synthesized with (13)C-enriched Ala or Pro, at the N-terminal or C-terminal side of the peptide, and the intermolecular PREs were measured. The results provide compelling evidence that the FAK-derived peptide binds the SH3 domain in two orientations. In the major state, the SH3 domain binds the peptide in the N-C orientation, whereas 20% of the time, the peptide binds in the C-N orientation. We conclude that the distinction between N-C and C-N orientations, which is based on crystal structures, might be artificial. The pseudosymmetric nature of the polyproline helix might allow for binding in both orientations in the solution state.
authors.affiliation: Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Bharat
authors.initials: B
authors.lastname: Somireddy Venkata
authors.affiliation: Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Peter H J
authors.initials: PH
authors.lastname: Keizers
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center (LUMC) , P.O. Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Jan W
authors.initials: JW
authors.lastname: Drijfhout
authors.affiliation: Leiden Institute of Chemistry, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D018909:src Homology Domains
chemical_list: D008024:Ligands; D010455:Peptides; D051416:Focal Adhesion Protein-Tyrosine Kinases