Analysis of the interaction of peptide-class II MHC molecule by photoaffinity labeling

The interaction between peptide and MHC class II molecule under SDSPAGE revealed two kinds of complexes:those in which the peptide dissociates from the class II molecule (unstable complexes) and those in which the peptide remains bound to the ab dimer (stable complexes).We have examined the interaction between peptides and the Ak molecule using photoaffinity probes.Peptides were conjugated on their N-terminus to 1251-azido salicylic acid (IASA), incubated with purified Ak, and then crosslinked.The complexes were analyzed by SDS-PAGE. Peptides, mostly derived from HEL, were selected on the basis of their binding affinities and S D S- s t ability. The natural peptide 48-61 has a critical anchor residue (Asp at 52 position) that is responsible both for high affinity and SDS-stability . The interaction of such IASA-48-61 with Ak molecule results in a stable complex that is poorly crosslinked.When the anchor residue was changed so to produce lower affinity binding and lower percentage of stable complexes, two sets of complexes were found : one was highly crosslinked (corresponding to the SDS-unstable complex) while another was poorly crosslinked, corresponding to the SDS-stable complex. Depending on the anchorage residue at the 52 position, the peptides were selectively crosslinked to the a, b or both chains of the Ak.Our results indicate two states of interaction of peptides with Ak. In one the peptide binds with high affinity and is highly constrained in the complex.In the second the peptide binds with lesser affinity and the Ak bound peptide has more degrees of freedom and adopts more than one conformation.