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Christopher Lee^*, Michael N. Liang^*, Keri M. Tate, Joshua D. Rabinowitz^*, Craig Beeson^*, Patricia P. Jones, and Harden M. McConnell^*

From the ^* Department of Chemistry and Department of Biological Sciences, Stanford University, Stanford, California 94305-5080

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.

Christopher Lee's present address is Department of Chemistry & Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095. Michael N. Liang's present address is Department of Chemistry, Harvard University, Cambridge, MA 02138. Keri M. Tate's present address is Anergen Inc., Redwood City, CA 94603. Craig Beeson's present address is Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700.

Abbreviations used: CLIP, class II–associated invariant chain peptide; D-ala, D-alanine; DM, dodecyl maltoside; EAE, experimental autoimmune encephalomyelitis; HPSEC, high-performance size exclusion chromatography; MBP, myelin basic protein.

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