Novel mode of ligand binding by the SH2 domain of the human XLP disease gene product SAP/SH2D1A

Background: The proteins homology 2 (SH2) domains of cytoplasmic signaling proteins generally bind phosphotyrosine (pTyr) sites in the context of carb oxyterminal residues. SAP (also known as SH2D1A or DSHP), the product of th e gene that is mutated in human X-linked lymphoproliferative (XLP) disease, comprises almost exclusively a single SH2 domain, which may modulate T-cel l signaling by engaging T-cell co-activators such as SLAM, thereby blocking binding of other signaling proteins that contain SH2 domains, The SAP-SLAM interaction can occur in a phosphorylation-independent manner. Results: To characterize the interaction between SAP and SLAM, we synthesiz ed peptides corresponding to the SAP-binding site at residue Y281 in SLAM. Both phosphorylated and non-phosphorylated versions of an 11-residue SLAM p eptide bound SAP, with dissociation constants of 150 nM and 330 nM, respect ively. SLAM phosphopeptides that were truncated either at the amino or carb oxyl terminus bound with high affinity to SAP, suggesting that the SAP SH2 domain recognizes both amino-terminal and carboxy-terminal sequences relati ve to the pTyr residue. These results were confirmed by nuclear magnetic re sonance (NMR) studies on N-15- and C-13-labeled SAP complexed with three SL AM peptides: an amino-terminally truncated phosphopeptide, a carboxy-termin ally truncated phosphopeptide and a non-phosphorylated Tyr-containing full- length peptide. Conclusions: The SAP SH2 domain has a unique specificity. Not only does it bind peptides in a phosphorylation-independent manner, it also recognizes a pTyr residue either preceded by amino-terminal residues or followed by car boxy-terminal residues. We propose that the three 'prongs' of a peptide lig and (the amino and carboxyl termini and the pTyr) can engage the SAP SH2 do main, accounting for its unusual properties. These data point to the flexib ility of modular protein-interaction domains.