Circular dichroism determination of class I MHC‐peptide equilibrium dissociation constants

Class I major histocompatibility complex (MHC) molecules bind short peptides derived from proteins synthesized within the cell. These complexes of peptide and class I MHC (pMHC) are transported from the endoplasmic reticulum to the cell surface. If a clonotypic T cell receptor expressed on a circulating T cell binds to the pMHC complex, the cell presenting the pMHC is killed. In this manner, some tumor cells expressing aberrant proteins are recognized and removed by the immune system. However, not all tumors are recognized efficiently. One reason hypothesized for poor T cell recognition of tumor-associated peptides is poor binding of those peptides to class I MHC molecules. Many peptides, derived from the proto-oncogene HER-2/neu have been shown to be recognized by cytotoxic T cells derived from HLA-A2؉ patients with breast cancer and other adenocarcinomas. Seven of these peptides were found to bind with intermediate to poor affinity. In particular, GP2 (HER-2/neu residues 654 -662) binds very poorly even though it is predicted to bind well based upon the presence of the correct HLA-A2.1 peptide-binding motif. Altering the anchor residues to those most favored by HLA-A2.1 did not significantly improve binding affinity. The crystallographic structure shows that unlike other class I-peptide structures, the center of the peptide does not assume one specific conformation and does not make stabilizing contacts with the peptide-binding cleft.

Section: Peptidementioning

confidence: 54%

Section: Peptidementioning

confidence: 99%

Poor Binding of a HER-2/neu Epitope (GP2) to HLA-A2.1 Is due to a Lack of Interactions with the Center of the Peptide

Kuhns¹,

Batalia²,

Yan³

et al. 1999

“…T Cell Assays-The CD8 ϩ CTL clones RS56 (expressing the A6 TCR) (27), 10B7 (expressing the B7 TCR) (27), and 1E7 (not previously described), all specific for human T cell lymphotrophic virus, type I (HTLV-I) Tax [11][12][13][14][15][16][17][18][19] -HLA-A2, were assayed on HLA-A2 wild-type and Lys 66 mutant-transfected Hmy2.C1R cells (28). The gene usage and CDR loop sequences of the TCRs expressed by these clones are shown in Table I (1E7 expresses two productively rearranged ␣ chains).…”

Section: Methodsmentioning

confidence: 99%

“…We previously used circular dichroism to monitor the thermal unfolding of the wild-type and the R65A and K66A mutant peptide-MHC molecules (4). Because of the linkage between peptide binding and protein stability, thermal unfolding is frequently used as a probe of peptide binding affinity (14). As neither the R65A nor the K66A mutation resulted in a significant change in the apparent T m of the peptide-MHC complex, we concluded that the effects seen with the R65A and K66A mutations were not likely due to weaker binding of the peptide to the mutant MHC molecules.…”

mentioning

confidence: 89%

Strategic Mutations in the Class I Major Histocompatibility Complex HLA-A2 Independently Affect Both Peptide Binding and T Cell Receptor Recognition

Baxter¹,

Gagnon

Davis-Harrison³

et al. 2004

Mutational studies of T cell receptor (TCR) contact residues on the surface of the human class I major histocompatibility complex (MHC) molecule HLA-A2 have identified a "functional hot spot" that comprises Arg 65 and Lys 66 and is involved in recognition by most peptide-specific HLA-A2-restricted TCRs. Although there is a significant amount of functional data on the effects of mutations at these positions, there is comparatively little biochemical information that could illuminate their mode of action. Here, we have used a combination of fluorescence anisotropy, functional assays, and Biacore binding experiments to examine the effects of mutations at these positions on the peptide-MHC interaction and TCR recognition. The results indicate that mutations at both position 65 and position 66 influence peptide binding by HLA-A2 to various extents. In particular, mutations at position 66 result in significantly increased peptide dissociation rates. However, these effects are independent of their effects on TCR recognition, and the Arg 65 -Lys 66 region thus represents a true "hot spot" for TCR recognition. We also made the observation that in vitro T cell reactivity does not scale with the half-life of the peptide-MHC complex, as is often assumed. Finally, position 66 is implicated in the "dual recognition" of both peptide and TCR, emphasizing the multiple roles of the class I MHC peptide-binding domain.Recognition of a peptide bound to a major histocompatibility complex protein (peptide-MHC) 1 by the ␣␤ T cell receptor (TCR) is necessary for the initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. TCRs bind peptide-MHC in a diagonal-to-orthogonal fashion, interacting with elements of both the peptide and the MHC (1-3). Numerous studies have probed the interfaces between TCRs and their ligands through the use of altered peptides or site-directed mutagenesis of the MHC (e.g. see . These studies have been useful in identifying hot spots within individual interfaces, as well as predicting the biophysical mechanisms by which T cell receptors bind their ligand (9).In our studies of TCR recognition of the human class I MHC HLA-A*0201 (referred to as HLA-A2) presenting the Tax peptide (sequence LLFGYPVYV; see Ref. 10), we identified a functional hot spot consisting of arginine 65 and lysine 66 on the HLA-A2 ␣1 helix (4). In a mutagenesis experiment involving 15 HLA-A2 amino acids contacted by the Tax-HLA-A2-specific ␣␤ T cell receptor A6, only two mutations, Arg 65 3 Ala (R65A) and Lys 66 3 Ala (K66A), resulted in significantly reduced T cell effector functions when the mutants were used to present the Tax peptide to T cells expressing the A6 receptor. Similar results were found with T cells expressing a different Tax-HLA-A2-specific TCR, B7. Lys 66 was also identified as a critical position influencing T cell reactivity in at least one other mutagenesis study of HLA-A2 (8).The general nature of the Arg 65 -Lys 66 hot spot in the recognition of Tax...

“…Data were also collected from "empty" HLA-E molecules refolded in the absence of added peptides. Prior studies have shown that classical MHC class I proteins (H-2K d ) have T m values in the range of 52-61°C depending upon the peptide used, whereas "empty" molecules have a T m of 45°C (36,37). In these experiments, the HLA-G-and HLA-B7-derived peptides yield identical T m values when bound by either HLA-E G (T m ϭ 52°C) or HLA-E R (T m ϭ 49°C), suggesting that these substitutions at positions P7 and P8 do not affect stability.…”

Section: Thermal Stability Of Hla-e In Complex With Different Peptidementioning

confidence: 99%

HLA-E Allelic Variants

Strong¹,

Holmes²,

Li³

et al. 2003

260

114

Previous studies of HLA-E allelic polymorphism have indicated that balancing selection may be acting to maintain two major alleles in most populations, indicating that a functional difference may exist between the alleles. The alleles differ at only one amino acid position, where an arginine at position 107 in HLA-E*0101 (E R ) is replaced by a glycine in HLA-E*0103 (E G ). To investigate possible functional differences, we have undertaken a study of the physical and biochemical properties of these two proteins. By comparing expression levels, we found that whereas steady-state protein levels were similar, the two alleles did in fact differ with respect to cell surface levels. To help explain this difference, we undertook studies of the relative differences in peptide affinity, complex stability, and three-dimensional structure between the alleles. The crystal structures for HLA-E G complexed with two distinct peptides were determined, and both were compared with the HLA-E R structure. No significant differences in the structure of HLA-E were induced as a result of binding different peptides or by the allelic substitution at position 107. However, there were clear differences in the relative affinity for peptide of each heavy chain, which correlated with and may be explained by differences between their thermal stabilities. These differences were completely consistent with the relative levels of the HLA-E alleles on the cell surface and may indeed correlate with functional differences. This in turn may help explain the apparent balancing selection acting on this locus.