Interaction between the N-terminal SH3 domain of Nckα and CD3ɛ-derived peptides: Non-canonical and canonical recognition motifs

The first SH3 domain (SH3.1) of Nckα specifically recognizes the proline-rich region of CD3ɛ, a subunit of the T cell receptor complex. We have solved the NMR structure of Nckα SH3.1 that shows the characteristic SH3 fold consisting of two antiparallel β-sheets tightly packed against each other. According to chemical shift mapping analysis, a peptide encompassing residues 150–166 of CD3ɛ binds at the canonical SH3 binding site. An exhaustive comparison with the structures of other SH3 domains able and unable to bind CD3ɛ reveals that Nckα SH3.1 recognises a non-canonical PxxPxxDY motif that orientates at the binding site as a class II ligand. A positively charged residue (K/R) at position − 2 relative to the WW sequence at the beginning of strand β3 is crucial for PxxDY recognition. A 14-mer optimised Nckα SH3.1 ligand was found using a multi-substitution approach. Based on NMR data, this improved ligand binds Nckα SH3.1 through a PxxPxRDY motif that combines specific stabilising interactions corresponding to both canonical class II, PxxPx(K/R), and non-canonical PxxPxxDY motifs. This explains its higher capacity for Nckα SH3.1 binding relative to the wild type sequence.