Beeson Group Research

Reactions of Peptides and Class II MHC Proteins

Antigen presenting cells process antigen protein into peptide fragments that are subsequently presented to T cells. Usually T cells are tolerant of MHC proteins presenting self peptides. In collaboration with Joan Goverman in the Department of Immunology, we are studying breakdown in T-cell tolerance to a region of the myelin basic protein involved in a mouse model for Multiple Sclerosis. We are using molecular modeling, peptide mutations and kinetic measurements to examine the binding of this peptide to its MHC protein and the subsequent T-cell response. We have recently demonstrated that there are two distinct regions within this sequence that are presented to the T-cells and that the recognition is degenerate (isomeric) among some of these T cells. Interestingly, these two epitopes do not present homologous amino acid residues to the T cell receptor (TCR). We have also found a correlation between tolerance to these peptides and the kinetic stability of the complexes. This is the first study of its kind to examine the role of peptide-MHC isomers in an autoimmune disease.

Highly Cross-reactive T Cell Responses to Myelin Basic Protein Epitopes
We have identified two, non-overlapping epitopes in myelin basic protein presented by I-A^u that are responsible for tolerance induction to this self antigen. A large number of T cells expressing diverse TCRs were strongly cross-reactive to both epitopes. Shown are the two core peptides that bind to I-A^u and are presented to T cells. Residues P2, P3, P5, and P8 are directed to the TCR. Residues P1, P4, P6, and P9 are MHC binding contacts. It was found that the TCR-contact residues in each peptide are highly dissimilar and are only recognized within the context of the other amino acids in that peptide's sequence. This observation indicates that both buried and exposed residues of each peptide contribute to the sculpting of distinct antigenic surfaces. Each surface is sufficiently unique that functional TCR contacts cannot be interchanged between the two epitopes. Together these results indicate that predicting cross-reactive peptides by defining TCR contacts on a single epitope is likely to under-represent the complete repertoire of cross-reactive epitopes.

In collaboration with Andrea Sant at the University of Chicago we are also studying how mutations in the structure of the MHC affect antigen presentation. We are using mutations of the MHC protein and peptide dissociation kinetics to elaborate the relative energetics of different binding interactions. We've shown that a single hydrogen bond from the I-A^d MHC b-His81 side-chain to peptide backbone is a major binding contact for many peptides. The sensitivities to this mutation range from factors of 5- to 200-fold and are inversely correlated with kinetic stability. These results demonstrate how peptide binding is stabilized by a complex range of hydrogen bond and pocket interactions. We've also shown that the rate determining step affected by this hydrogen bond is not affected by pH. A second rate determing step is affected by pH. These results demonstrate for the first time that dissociation of many peptides from the I-A^d MHC protein proceeds via a common kinetic intermediate.

Ova(323-334) peptide bound to the MHC protein I-A^d
The peptide is bound in a groove formed by the helices of the a-chain (yellow) and b-chain (green). Binding is stabilized by hydrogen bonds between protein side-chains and the peptide backbone (not shown). There are also binding interactions between peptide side-chains and pockets within the MHC peptide-binding groove. The b-His-81 residue (highlighted in blue) forms a hydrogen bond to a peptide carbonyl proximal to the P1 pocket. Mutation of b-His-81 to an Asn eliminates this hydrogen bond.

The Ovalbumin peptide binds to the MHC protein using two functionally distinct registers
Previous studies have shown that most of the residues required for binding of the chicken ovalbumin (Ova) 323-339 peptide to the I-A^d MHC class II protein are contained within the shorter 325-336 peptide. This observation is somewhat inconsistent with the x-ray structure of the Ova peptide covalently attached to I-A^d in which residues 323 and 324 form binding interactions with the protein (top). A second register for the Ova(325-336) peptide was found where residues 326 and 327 occupy positions similar to residues 323 and 324 in the 1IAO structure (bottom). The Ova(323-335) peptide that binds in the 1IAO register does not stimulate a T-cell hybridoma that is stimulated by Ova(325-336) bound in the alternate register. These results demonstrate that a single peptide can bind to an MHC peptide in alternate registers producing distinct T-cell responses.

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