Study of the interaction of the medium chain mu 2 subunit of the clathrin-associated adapter protein complex 2 with cytotoxic T-lymphocyte antigen 4 and CD28

The medium chain mu2 subunit (AP50) of the clathrin-associated adapter prot ein complex 2 (AP-2) interacts specifically with the tyrosine-based signals of several integral membrane proteins through the consensus sequence YXX P hi, where X can be any residue and Phi is a large hydrophobic residue. Usin g surface plasmon resonance combined with structural information, we have a nalysed the interaction of AP50 with peptides derived from the cytoplasmic tail of cytotoxic T-lymphocyte antigen 4 (CTLA-4). The crystal structure of AP50 in,complex with a CTLA-4-derived peptide was determined to 3.6 Angstr om (1 Angstrom = 0. 1 nm) resolution. The binding domain of AP50 (residues 164-435) was expressed in Escherichia coli and purified. In agreement with previous reports, the AP50 domain bound to residues 152-174 of CTLA-4, but not to the same peptide that was phosphorylated at the single tyrosine resi due (position 165). The interaction exhibited fast kinetics with rapid on a nd off rates and a K-d of 0.7 muM. In order to further understand why AP50 binds to CTLA-4, but not to the homologous receptor CD28, a comparison of b inding of AP50 with five peptides with single changes in and around the YXX Phi motif to the equivalent residues of CD28 was made. T162H greatly reduc ed binding, whereas T161L had little effect. Mutations G163S, V164D and K16 7N all exhibited reduced binding. Modelling of the single amino acid change s using structural information, was in broad agreement with the binding dat a, demonstrating that residues outside of the YXX Phi motif are also import ant in the interaction of membrane proteins with AP50.