EVIDENCE THAT THE AUTOIMMUNE ANTIGEN MYELIN BASIC-PROTEIN (MBP) AC1-9BINDS TOWARDS ONE END OF THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) CLEFT

The NH2-terminal peptide of myelin basic protein (MBP) bound to the cl ass II major histocompatibility complex (MHC) protein I-A(u) is an imm unodominant epitope in experimental autoimmune encephalomyelitis, a mu rine model of multiple sclerosis. However, the MBP-I-A(u) complex is v ery unstable. To investigate this, we performed site-directed mutagene sis of the I-A(u) MHC protein and the MBP peptide. Biochemical, T cell activation, and molecular modeling studies of mutant complexes demons trate that the MBP peptide's key residue for MHC binding, lysine 4, is buried in the P6 pocket of I-A(u), which is predominantly hydrophobic . This implies that the MBP-I-A(u) complex differs from more stable co mplexes in two respects: (a) the peptide leaves the NH2-terminal regio n of the MHC peptide-binding cleft unoccupied; (b) the peptide is not anchored by typical favorable interactions between peptide side chains and MHC pockets. To test these hypotheses, a modified MBP peptide was designed based on molecular modeling, with the aim of producing stron g I-A(u) binding. Extension of the NH, terminus of MBP with six amino acids from the ova peptide, and replacement of the lysine side chain i n the P6 pocket with an aromatic anchor, results in >1,000-fold increa sed binding stability. These results provide an explanation for the un usual peptide-MHC-binding kinetics of MBP, and should facilitate an un derstanding of why mice are not tolerant to this self-peptide-MHC comp lex.