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A Structural Difference Limited to One Residue of the Antigenic Peptide Can Profoundly Alter the Biological Outcome of the TCR-Peptide/MHC Class I Interaction¹

Cole T. Thomson^*, Alexis M. Kalergis, James C. Sacchettini and Stanley G. Nathenson^2,,

Departments of ^* Biochemistry, Microbiology and Immunology, and Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461; and Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843

	Abstract

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The vesicular stomatitis virus (VSV) octapeptide RGYVYQGL binds to H-2K^b and triggers a cytotoxic T cell response in mice. A variant peptide, RGYVYEGL (E6) with a glutamic acid for glutamine replacement at position 6 of the VSV peptide, elicits a T cell response with features that are quite different from those elicited by the wild-type VSV peptide. The differences found in the nature of the T cells responding to the E6 peptide include changes in both the V elements and the sequences of the complementarity-determining region 3 loops of their TCRs. Further experiments found that the E6 peptide can act as an antagonist for VSV-specific T cell hybridomas. To determine whether these differences in V usage, complementarity-determining region 3 sequences, and the switch from agonism to antagonism are caused by a conformational change on the MHC, the peptide, or both, we determined the crystal structure of the variant E6 peptide bound to H-2K^b. This structure shows that the only significant structural difference between H-2K^b/E6 and the previously determined H-2K^b/VSV is limited to the side chain of position 6 of the peptide, with no differences in the MHC molecule. Thus, a minor conformational change in the peptide can profoundly alter the biological outcome of the TCR-peptide/MHC interaction.

	Introduction

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In an attempt to explore the structural basis of Ag recognition by cytotoxic T cells, recent studies have focused on an increasingly detailed examination of the interactions between TCR and peptide/MHC class I (pMHC)³ complex, including those comprised of altered peptide ligands (1, 2, 3). Using the H-2K^b/vesicular stomatitis virus (VSV) complex as a model system, we have explored the biological effect of amino acid substitutions of both peptide and MHC residues that are likely to interface with the TCR. We have shown that the QE substitution at position 6 (E6) of the naturally occurring VSV peptide, can profoundly alter, both in vitro and in vivo, T cell response. Analysis of the TCR-pMHC binding patterns of a set of T cell clones specific for the VSV peptide showed that the QE substitution alters the interface of TCR binding to the MHC compared with the wild-type VSV peptide (4). Further, using mice with a transgenic TCR -chain, Wang et al. (5) have shown that immunization with E6 elicits T cells with TCRs containing different complementarity-determining region (CDR)-3 motifs than the TCRs on T cells arising after immunization with VSV peptide. In addition, when the V element usage of these peptide-specific T cell populations was analyzed, it was found that E6 peptide immunization induced a different TCR V usage than that found in VSV peptide-immunized mice (6). Thus, the QE substitution at position 6 of the peptide results in significant differences in the nature of the TCRs of the responding T cell populations. In addition to these findings, we have recently observed that the E6 peptide is able to antagonize an H-2K^b/VSV-specific TCR (N30.7), indicating that this alteration in the VSV peptide sequence is enough to inhibit the recognition of the wild-type peptide by this particular TCR (7).

Given the significant biological effects of the QE substitution on the TCR structure and function, we decided to investigate whether these functional alterations in the T cell responses were due to structural changes on the heavy chain of the H-2K^b molecule induced by this peptide variant. With this aim, we have determined the crystal structure of the H-2K^b/E6 complex and compared it with the H-2K^b/VSV structure. We found that the QE substitution at position 6 of VSV peptide resulted in structural changes limited to the peptide and that did not involve the heavy chain of the MHC molecule. Thus, subtle conformational changes in the ligand can cause profound alterations in the type of T cell response triggered by the TCR-pMHC interaction.

	Materials and Methods

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Synthetic peptide production

E6 (RGYVYEGL) was synthesized by the Peptide Synthesis Facility of the Albert Einstein College of Medicine. The peptide was purified to >98% homogeneity by reversed-phase HPLC and the identity of the peptide was confirmed by mass spectrometry.

Production, folding, and crystallization of H-2K^b/E6 complex

As previously described by Zhang et al. (8), transformed BL21 (DE3)pLysS cells were grown in Luria-Bertani medium, lysed with a French press, and then processed by several cycles of centrifugation and washing, resulting in >95% pure inclusion bodies. The H-2K^b and ₂-microglobulin (₂m) inclusion bodies were solubilized in a buffered urea solution. Peptide, H-2K^b, and ₂m were mixed in a molar ratio of 10:1:1 at an overall concentration of 0.3 mg/ml; the mixture was dialyzed against aqueous buffer in 500 MWCO dialysis. After dialysis, the material was concentrated and then purified on an ion exchange column. The homogeneity and molecular mass of the complex was assayed by gel exclusion chromatography (8, 9). Conditions for crystallization were similar to those previously established by Zhang et al. (8): using the hanging drop vapor diffusion method with a reservoir buffer of 0.1 M calcium acetate/0.1 M cacodylate buffer (pH 6.5)/15–20% and polyethylene glycol 8000.

Data collection, molecular replacement, and refinement

X-ray data was collected at the X-9B beamline at Brookhaven National Laboratories Synchrotron Light Source on a Mar345 image plate system using a single crystal at -165°C (Brookhaven Instruments, Holtsville, NY). Data were processed with Denzo and Scalepack. The crystal belongs to the space group P2₁ (unit cell a = 89.3 Å, b = 82.3 Å, c = 66.4 Å and = 111.0°) containing two pMHC complexes per asymmetric unit. See Table I for dataset statistics.

X-Plor version 3.851 (12) was used to refine the structure through multiple cycles of model building. A random set of 5% of reflections were sequestered from further refinement and used for calculation of R_free (1733 reflections for data to 2.3 Å). Bulk solvent corrections were applied to the Fo throughout the refinement (13). Data were gradually extended to 100–2.3 Å with a F/ cutoff of 2.0, and individual atomic B factors were used at the end of the refinement process. Areas with poor geometry were fit using simulated annealing omit maps. Although clear electron density for peptide could be seen from the initial stages of model building, peptide residues were not included until the final stages of refinement.

Because the Fo was noted to be somewhat anisotropic, an overall anisotropic B factor correction (B₁₁ = 21.094, B₂₂ = 9.853, B₃₃ = 21.044) was applied, resulting in a reduction of the R_free by 1.5% and a significant improvement in electron density maps. The crystal form has two pMHC complexes per asymmetric unit; these complexes are quite similar, with C carbon RMS deviation for the Ag presentation domains of 0.61 Å. RMS deviation between the two peptides is 0.377 Å. There was no use of noncrystallographic restraints during any part of the refinement. One complex contains a peptide with a higher average B factor than the other (71 Ų vs 46 Ų). Average B factors for the two heavy chains are more similar (53 Å vs 59 Å). Differences in average peptide B factor may be due to crystal packing, but omit electron density maps (calculated from randomized coordinates to reduce model bias) of both peptides are of high quality.

	Results and Discussion

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Residue 6 of the peptide is positioned differently in H-2K^b/VSV and in H-2K^b/E6

To evaluate the structural changes that are responsible for the different biological outcomes that result from the Q to E substitution at position 6 of the VSV peptide, H-2K^b/₂m/E6 was folded, crystallized, and its structure was determined to 2.3 Å by x-ray crystallography. This crystal structure was compared with the previously reported H-2K^b/VSV structure with the same space group (10), demonstrating that the glutamic acid at position 6 of the E6 peptide (Fig. 1, A and B) is positioned differently in the cleft than the glutamine of position 6 in the VSV wild type. The glutamic acid side chain of the E6 peptide is 1.5 Å closer to the 1 helix than the position 6 glutamine of the wild-type VSV peptide; this orientation is conserved in both complexes of the asymmetric unit (see Fig. 1, C and D). Although shifted, position 6 of E6 remains solvent exposed and a likely TCR contact residue. This structural difference is attributable to the hydrogen bond observed between the NE2 atom of the Q6 residue and the glutamic acid at position 152 on the 2 helix of the H-2K^b molecule. The negatively charged side chain at position 6 of the E6 peptide no longer hydrogen bonds to the E152 residue of H-2K^b (Fig. 1, C and D). The position and conformation of other nearby residues in the peptide and the Ag-binding groove are not significantly altered by the QE replacement, including the position of E152. In both the H-2K^b/E6 and H-2K^b/VSV complexes, the carboxylate of E152 forms a salt bridge with the positively charged side chain of R155, a nearby amino acid on the 2 helix; this may account for the relative immobility of residue 152 compared with the position 6 of the peptide. Thus, the only significant conformational difference between the two structures attributable to the QE substitution is limited to position 6 of the peptide. No other structural differences attributable to the substituted amino acids are noted. The QE replacement does alter the electrostatic surface of the molecule, and this electrostatic change is likely the only other significant difference between the two structures.

View larger version (49K): [in this window] [in a new window]   FIGURE 1. Location and orientation of side chains at position 6 of VSV and E6 peptides bound to H-2Kb. Top-down (A) and side (B) views of TCR-binding region of the H-2Kb/E6 complex. -carbon trace of heavy chain in blue, peptide trace in green. Glutamic acid side chains at position 6 of E6 peptide and position 152 of H-2Kb are shown in red and yellow, respectively. C and D, Shake Fo-Fc omit maps at 2.5 of position 6 side chain of the E6 peptide from both asymmetric units. H-2Kb/E6 and H-2Kb/VSV structures have been superimposed. Note the hydrogen bond between the E152 side chain (at right) and the N of the Q6 side chain (center). E6 side chain is positioned 1.5 Å further from E152 than Q6 of the VSV peptide. C is yellow; N, blue; O, red.

To further evaluate the role of the antigenic peptide structure in controlling the nature of the responding TCR repertoire, the V family usage in CD8⁺ T cell populations obtained from TCR- transgenic mice immunized with VSV or E6 peptide was analyzed (6). Thus, in VSV peptide-immunized mice, the predominant TCR V gene element in CD8⁺ CTL populations was V13 (80%) (Fig. 2A). This V element represented <5% of CD8⁺ T cells obtained from naive TCR- transgenic mice (p < 0.005). No significant expansion of any other V element was observed (6). However, when TCR- transgenic mice were immunized with the E6 peptide variant, the predominant V gene element expressed by CD8⁺ T cells was V7 (80%) (Fig. 2A). This V family was significantly expanded compared both with naive and VSV-immunized mice (p < 0.0005). The alteration in the TCR V usage caused by the Q to E substitution at position 6 of VSV could be the result of a direct interaction between that position of the peptide and the germline-encoded CDR loops of the TCRs from the T cell clones expanded after peptide immunization. This is noteworthy, because Ag recognition has been perceived as a CDR3-mediated event, whereas the germline-encoded CDR1 and CDR2 loops have been considered as being important for the MHC restriction aspect of recognition (14). However, the crystal structure of TCR-pMHC complexes have shown that both CDR1 and CDR1 are likely to contact residues on the N- and C-terminal half of the peptide, respectively, indicating that CDR1 loops could be involved in the recognition of specific peptides (15, 16, 17). Because our data indicate that the QE substitution affects only the peptide bound to the H-2K^b groove, it seems unlikely that the heavy chain of the H-2K^b molecule could play a role in the V usage changes induced by the Q to E substitution at position 6 of VSV. This observation supports the notion that the V usage change caused by the QE replacement would be necessitated by an interaction between the position 6 side chain of the E6 peptide and the TCR CDR1 loop of the responding CD8⁺ T cell (6).

View larger version (13K): [in this window] [in a new window]   FIGURE 2. A, V family usage in TCR- transgenic mice after peptide immunization. E6 immunization expands CD8+ T cells expressing the V7 element, while immunization with the wild-type VSV peptide results in a predominant expansion of CD8+ T cells expressing the V13 element (*, p < 0.005, **, p < 0.0005). B, E6 peptide specifically antagonizes the activation of T cell hybridomas expressing the H-2Kb/VSV-specific N30.7 TCR. As control, hybridomas expressing the H-2Kb/VSV-specific N15 TCR were included in the experiments. Antagonism was evaluated by Ag prepulse assays and data are expressed as percentage of inhibition of IL-2 release compared with the amount of IL-2 induced in absence of the E6 peptide. As H-2Kb-competition control, the Sendai virus nucleoprotein-derived peptide (FAPGNYPAL) was also tested for antagonism of N30.7 and N15 TCR. The V family usage data were obtained from reference 6 and the TCR antagonism data were obtained from Ref. 7 .

Our observation that the E6 peptide antagonizes the H-2K^b/VSV-specific N30.7 TCR indicates that structural changes limited to a single position of the antigenic peptide are sufficient to cause TCR antagonism. Two previous studies in which the structures of MHC molecules complexed with antagonist peptides carrying single amino acid replacements were compared with the wild-type agonist peptide showed minor structural changes in MHC residues (18, 19, 20, 21). In agreement with our findings, those studies indicated that alteration of TCR contact residues results in only a small conformational change of the TCR-binding surface of the pMHC complex. In addition to providing evidence that TCR antagonism can be caused by limited changes in conformation and charge of peptide residues that contact the TCR, our study indicates that these minor conformational changes could also alter the TCR repertoire of the entire CD8⁺ T cell population in response to peptide immunization. Moreover, the limited conformational changes on the TCR-binding surface of the pMHC complex caused by the QE substitution of position 6 of the VSV peptide also support the notion that alterations in the recognition contacts of T cell clones (4, 22) caused by single amino acid replacements in the antigenic peptide are unlikely to be due to a major structural rearrangements of the pMHC complex.

Considered together, the biological and structural observations presented here indicate that subtle structural changes in the pMHC complex can lead to profound changes in the biological response of T cells. The molecular understanding of the interactions controlling the T cell response is important for the design of strategies aimed to manipulating immune responses.

	Acknowledgments

 
We thank Drs. Steve Almo, Anne Davidson, Betty Diamond, and Matthew Scharff, as well as Matthew Roden for critical reading of the manuscript. We also thank Edith Palmieri for folding and purification of H-2K^b/E6 and Marie Muranelli for secretarial assistance.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants RO1 AI07289-32, 5T52CA09173-23, RO1 AR42533-5, RO1 AI41240, and The Welch Foundation. The construction and operation of beamline X9B is supported by the Biotechnology Research Resource program of the National Institute of Health, Grant P41-RR01633. The National Synchrotron Light Source is supported by the Department of Energy, Division of Materials Sciences, Office of Energy Research.

² Address correspondence and reprint requests to Dr. Stanley G. Nathenson, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Chanin Building 407, Bronx, NY 10461.

³ Abbreviations used in this paper: pMHC, peptide/MHC class I; VSV, vesicular stomatitis virus; CDR, complementarity-determining region; ₂m, ₂-microglobulin.

Received for publication November 6, 2000. Accepted for publication January 2, 2001.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1. Sloan-Lancaster, J., P. M. Allen. 1996. Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology. Annu. Rev. Immunol. 14:1.[Medline]
2. Collins, E. J., J. A. Frelinger. 1998. Altered peptide ligand design: altering immune responses to class I MHC/peptide complexes. Immunol. Rev. 163:151.[Medline]
3. Abrams, S. I., J. Schlom. 2000. Rational antigen modification as a strategy to upregulate or downregulate antigen recognition. Curr. Opin. Immunol. 12:85.[Medline]
4. Ono, T., T. P. DiLorenzo, F. Wang, A. M. Kalergis, S. G. Nathenson. 1998. Alterations in TCR-MHC contacts subsequent to cross-recognition of class I MHC and singly substituted peptide variants. J. Immunol. 161:5454.[Abstract/Free Full Text]
5. Wang, F., T. Ono, A. M. Kalergis, W. Zhang, T. P. DiLorenzo, K. Lim, S. G. Nathenson. 1998. On defining the rules for interactions between the T cell receptor and its ligand: a critical role for a specific amino acid residue of the T cell receptor -chain. Proc. Natl. Acad. Sci. USA 95:5217.[Abstract/Free Full Text]
6. Kalergis, A. M., T. Ono, F. Wang, T. P. DiLorenzo, S. Honda, S. G. Nathenson. 1999. Single amino acid replacements in an antigenic peptide are sufficient to alter the TCR V repertoire of the responding CD8⁺ cytotoxic lymphocyte population. J. Immunol. 162:7263.[Abstract/Free Full Text]
7. Kalergis, A. M., S. G. Nathenson. 2000. Altered peptide ligand-mediated TCR antagonism can be modulated by a change in a single amino acid residue within the CDR3 of an MHC I-restricted TCR. J. Immunol. 165:280.[Abstract/Free Full Text]
8. Zhang, W., A. C. Young, M. Imarai, S. G. Nathenson, J. C. Sacchettini. 1992. Crystal structure of the major histocompatibility complex class I H-2K^b molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition. Proc. Natl. Acad. Sci. USA 89:8403.[Abstract/Free Full Text]
9. Kalergis, A. M., E. C. Goyarts, E. Palmieri, S. Honda, W. Zhang, S. G. Nathenson. 2000. A simplified procedure for the preparation of MHC/peptide tetramers: chemical biotinylation of an unpaired cysteine engineered at the C-terminus of MHC-I. J. Immunol. Methods 234:61.[Medline]
10. Fremont, D. H., M. Matsumura, E. A. Stura, P. A. Peterson, I. A. Wilson. 1992. Crystal structure of two viral peptides in complex with murine MHC class I H-2K^b. Science 257:919.[Abstract/Free Full Text]
11. Navaza, J.. 1994. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50:157.
12. Weis, W. I., A. T. Brunger, J. J. Skehel, D. C. Wiley. 1990. Refinement of the influenza virus hemagglutinin by simulated annealing. J. Mol. Biol. 212:737.[Medline]
13. Jones, T. A., J. Y. Zou, S. W. Cowan, M. Kjeldgaard. 1991. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47:110.
14. Davis, M. M., J. J. Boniface, Z. Reich, D. Lyons, J. Hampl, B. Arden, Y. Chien. 1998. Ligand recognition by T cell receptors. Annu. Rev. Immunol. 16:523.[Medline]
15. Ding, Y. H., K. J. Smith, D. N. Garboczi, U. Utz, W. E. Biddison, D. C. Wiley. 1998. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Immunity 8:403.[Medline]
16. Garboczi, D. N., P. Ghosh, U. Utz, Q. R. Fan, W. E. Biddison, D. C. Wiley. 1996. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384:134.[Medline]
17. Garcia, K. C., M. Degano, L. R. Pease, M. Huang, P. A. Peterson, L. Teyton, I. A. Wilson. 1998. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science 279:1166.[Abstract/Free Full Text]
18. Reid, S. W., S. McAdam, K. J. Smith, P. Klenerman, C. A. O’Callaghan, K. Harlos, B. K. Jakobsen, A. J. McMichael, J. I. Bell, D. I. Stuart, E. Y. Jones. 1996. Antagonist HIV-1 Gag peptides induce structural changes in HLA B8. J. Exp. Med. 184:2279.[Abstract/Free Full Text]
19. Ghendler, Y., M. K. Teng, J. H. Liu, T. Witte, J. H. Liu, K. S. Kim, P. Kern, H. C. Chang, J. H. Wang, E. L. Reinherz. 1998. Differential thymic selection outcomes stimulated by focal structural alteration in peptide/major histocompatibility complex ligands. Proc. Natl. Acad. Sci. USA 95:10061.[Abstract/Free Full Text]
20. Ding, Y. H., B. M. Baker, D. N. Garboczi, W. E. Biddison, D. C. Wiley. 1999. Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical. Immunity 11:45.[Medline]
21. Kirksey, T. J., R. R. Pogue-Caley, J. A. Frelinger, E. J. Collins. 1999. The structural basis for the increased immunogenicity of two HIV-reverse transcriptase peptide variant/class I major histocompatibility complexes. J. Biol. Chem. 274:37259.[Abstract/Free Full Text]
22. Ehrich, E. W., B. Devaux, E. P. Rock, J. L. Jorgensen, M. M. Davis, Y. H. Chien. 1993. T cell receptor interaction with peptide/major histocompatibility complex (MHC) and superantigen/MHC ligands is dominated by antigen. J. Exp. Med. 178:713.[Abstract/Free Full Text]

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