Evidence that the autoimmune antigen myelin basic protein (MBP) Ac1-9 binds towards one end of the major histocompatibility complex (MHC) cleft

The NH₂-terminal peptide of myelin basic protein (MBP) bound to the class II major histocompatibility complex (MHC) protein I-A(u) is an immunodominant epitope in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. However, the MBP-I-A(u) complex is very unstable. To investigate this, we performed site-directed mutagenesis of the I-A(u) MHC protein and the MBP peptide. Biochemical, T cell activation, and molecular modeling studies of mutant complexes demonstrate 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 complexes in two respects: (a) the peptide leaves the NH₂-terminal region 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 strong I-A(u) binding. Extension of the NH₂ terminus of MBP with six amino acids from the ova peptide, and replacement of the lysine side chain in the P6 pocket with an aromatic anchor, results in > 1,000-fold increased binding stability. These results provide an explanation for the unusual peptide-MHC-binding kinetics of MBP, and should facilitate an understanding of why mice are not tolerant to this self-peptide-MHC complex.