ANTIPEPTIDE ANTIBODIES DETECT CONFORMATIONAL-CHANGES OF THE INTER-SH2DOMAIN OF ZAP-70 DUE TO BINDING TO THE ZETA-CHAIN AND TO INTRAMOLECULAR INTERACTIONS

T cell receptor (TCR) triggering induces association of the protein ty rosine kinase ZAP-70, via its two src-homology 2 (SH2) domains, to di- phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (2pY-IT AMs) present in the intracellular tail of the TCR-zeta chain. The crys tal structure of the SH2 domains complexed with a 2pY-ITAM peptide sug gests that the 60-amino acid-long inter-SH2 spacer helps the SH2 domai ns 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 thi s region and probed ZAP-70 structure under various conditions. We show that the reactivity of antibodies directed at both sequences was dram atically augmented toward the tandem SH2 domains alone compared with t hat of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by s equences C-terminal to the SH2 domains, namely, the linker region and/ or the kinase domain, Moreover, antibody binding to the same two deter minants 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 conf iguration with the two SH2 domains near to each other.