title: Crystal structure analysis and solution studies of human Lck-SH3; zinc-induced homodimerization competes with the binding of proline-rich motifs.
abstract: In cytosolic Src-type tyrosine kinases the Src-type homology 3 (SH3) domain binds to an internal proline-rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src-type kinase Lck plays an important role during T-cell activation and development, since it phosphorylates the T-cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near-atomic resolution of 1.0 A. Unexpectedly, the Lck-SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc-binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline-rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck-SH3 domain also forms dimers in solution. The Zn(2+) dissociation constant from the Lck-SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline-rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc-induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck-SH3 homodimerization and binding of regulatory proline-rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc-binding site are highly conserved among Lck orthologues but not in other Src-type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T-cell activation.
authors.affiliation: Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Romir
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
authors.forename: Claudia
authors.initials: C
authors.lastname: Egerer-Sieber
authors.forename: Finn
authors.initials: F
authors.lastname: Bauer
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sticht
authors.forename: Yves A
authors.initials: YA
authors.lastname: Muller
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001667:Binding, Competitive / Q000187:drug effects*; D018360:Crystallography, X-Ray; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008024:Ligands; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011392:Proline / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D012996:Solutions; D012997:Solvents; D015032:Zinc / Q000494:pharmacology*; D018909:src Homology Domains
chemical_list: D008024:Ligands; D012996:Solutions; D012997:Solvents; D011392:Proline; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D015032:Zinc