MOLECULAR MODELING AND DESIGN OF INVARIANT CHAIN PEPTIDES WITH ALTERED DISSOCIATION KINETICS FROM CLASS-II MHC

We have used molecular modeling to design substitutions in an invarian t chain-derived peptide (CLIP), so as to alter the stability of its co mplex with class II major histocompatibility complex (MPIC) proteins. We sought first to test whether CLIP binds in the same way to differen t class II MHC proteins. We designed destabilizing substitutions of tw o residues (Met 91 and Met 99) previously predicted to act as the majo r anchor residues for binding to all class II MHC and measured their e ffect on CLIP's dissociation rate from a series of three murine I-A MH C proteins. Even a conservative substitution preserving size and hydro phobicity but reducing flexibility (leucine, a branched residue) cause d large accelerations in dissociation rates (up to 25-fold) at either position in all three MHC alleles, supporting the consistent role of t hese positions as the major anchors for MHC binding. These data also s upport the view that the special flexibility of the methionine side ch ains at these positions is essential for binding to diverse MHC molecu les. We also used molecular modeling to design allele-specific enhance ments of peptide binding. Designed substitutions of CLIP Pro 96 by Ala (for A(d)), Glu (A(k)), and Tyr (A(u)) each yielded strong enhancemen t of binding (up to 128-fold) for their targeted allele and only moder ate or destabilizing effects to the other alleles. These results demon strate the accuracy of the molecular models and the predictive value o f this modeling. Moreover, they provide strong evidence far the propos ed general model of invariant chain association, indicating that it bi nds to all class II MHC in the same conformation.