title: Interaction between the SH2 domains of ZAP-70 and the tyrosine-based activation motif 1 sequence of the zeta subunit of the T-cell receptor.
abstract: One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR zeta ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon zeta ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of zeta chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein-phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein-phosphopeptide complex relative to the protein alone.
authors.affiliation: Department of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Research and Development Center, Ridgefield, Connecticut 06877-0368, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: Labadia
authors.forename: S
authors.initials: S
authors.lastname: Jakes
authors.forename: C A
authors.initials: CA
authors.lastname: Grygon
authors.forename: D J
authors.initials: DJ
authors.lastname: Greenwood
authors.forename: J
authors.initials: J
authors.lastname: Schembri-King
authors.forename: S M
authors.initials: SM
authors.lastname: Lukas
authors.forename: T C
authors.initials: TC
authors.lastname: Warren
authors.forename: R H
authors.initials: RH
authors.lastname: Ingraham
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase
chemical_list: D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase