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

One of the key steps involved in T-cell activation is binding of the t yrosine kinase ZAP-70 via its two SH2 domains to peptide segments term ed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZA P-70 SH2 domains complexed to phosphopeptide revealed that the amino-t erminal phosphotyrosine-binding pocket is formed at the interface betw een the two SH2 domains. This study was designed to further characteri ze the binding between TCR zeta ITAM1 and the ZAP-70 SH2 domains as we ll as to assess the change in conformation of SH2 domain structure upo n 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 funct ional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH 2 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 ana lytical 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, indi cating a loss in solvent exposure for the tryptophan residues in the p rotein-phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein str ucture. Finally, thermal denaturation experiments showed an increase i n stability and cooperativity in unfolding for the protein-phosphopept ide complex relative to the protein alone. (C) 1997 Academic Press.