Analysis of immunoreceptor tyrosine‐based activation motif (ITAM) binding to ZAP‐70 by surface plasmon resonance

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Abstract
The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine‐based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six–eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP‐70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP‐70 to each TCR‐associated ITAM. First, we generated protein tyrosine phosphatase‐resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F2P) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase‐resistant analogs bind ZAP‐70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3ζ1 ITAM with a non‐peptidic linker made up of γ‐amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP‐70 SH2 domains.