title: Anti-peptide antibodies detect conformational changes of the inter-SH2 domain of ZAP-70 due to binding to the zeta chain and to intramolecular interactions.
abstract: T cell receptor (TCR) triggering induces association of the protein tyrosine kinase ZAP-70, via its two src-homology 2 (SH2) domains, to di-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (2pY-ITAMs) present in the intracellular tail of the TCR-zeta chain. The crystal structure of the SH2 domains complexed with a 2pY-ITAM peptide suggests that the 60-amino acid-long inter-SH2 spacer helps the SH2 domains to interact with each other to create the binding site for the 2pY-ITAM. To investigate whether the inter-SH2 spacer has additional roles in the whole ZAP-70, we raised antibodies against two peptides of this region and probed ZAP-70 structure under various conditions. We show that the reactivity of antibodies directed at both sequences was dramatically augmented toward the tandem SH2 domains alone compared with that of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by sequences C-terminal to the SH2 domains, namely, the linker region and/or the kinase domain. Moreover, antibody binding to the same two determinants was also inhibited when ZAP-70 or the SH2 domains bound to the zeta chain or to a 2pY-ITAM. Together, these two observations suggest a model in which intramolecular contacts keep ZAP-70 in a closed configuration with the two SH2 domains near to each other.
authors.affiliation: Molecular Immunology Unit, Department of Immunology, Pasteur Institut, 25, Rue du Docteur Roux, 75724 Paris Cedex 15, France.
authors.forename: L
authors.initials: L
authors.lastname: Grazioli
authors.forename: V
authors.initials: V
authors.lastname: Germain
authors.forename: A
authors.initials: A
authors.lastname: Weiss
authors.forename: O
authors.initials: O
authors.lastname: Acuto
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D008565:Membrane Proteins; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C054979:antigen T cell receptor, zeta chain; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human