Class I Major Histocompatibility Complex Anchor Substitutions Alter the Conformation of T Cell Receptor Contacts*

Ashwani K. SharmaDagger , Jennifer J. KuhnsDagger , Shuqin YanDagger , Randall H. FriedlineDagger , Brian LongDagger , Roland TischDagger §, and Edward J. CollinsDagger §||

From the Departments of Dagger  Microbiology and Immunology,  Biochemistry and Biophysics, and the § Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599

Received for publication, November 29, 2000, and in revised form, March 15, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

An immunogenic peptide (GP2) derived from HER-2/neu binds to HLA-A2.1 very poorly. Some altered-peptide ligands (APL) of GP2 have increased binding affinity and generate improved cytotoxic T lymphocyte recognition of GP2-presenting tumor cells, but most do not. Increases in binding affinity of single-substitution APL are not additive in double-substitution APL. A common first assumption about peptide binding to class I major histocompatibility complex is that each residue binds independently. In addition, immunologists interested in immunotherapy frequently assume that anchor substitutions do not affect T cell receptor contact residues. However, the crystal structures of two GP2 APL show that the central residues change position depending on the identity of the anchor residue(s). Thus, it is clear that subtle changes in the identity of anchor residues may have significant effects on the positions of the T cell receptor contact residues.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Class I major histocompatibility complex (MHC)1 proteins bind short peptides (8-11 amino acids) endogenously derived either from host or pathogen. These peptides bind to newly formed class I molecules in the endoplasmic reticulum. Peptide binding appears to be the final step in assembly of the complex (1). The complexes are presented to circulating T cells at the plasma membrane where clonotypic T cell receptors (TCR) on the surface of circulating cytotoxic T lymphocytes (CTL) may recognize the peptide-MHC complex (pMHC) and kill the presenting cell. An unaltered cell presents a population of self-peptides bound to class I MHC and is, for the most part, ignored by circulating T cells. However, cells infected by a virus or altered by neoplastic transformation present different peptides bound to class I MHC at the cell surface. These altered cells are recognized by clonotypic TCR on CD8+ T cells, and the T cells lyse the presenting cell. This action removes either the source of virus replication or the potential tumor (2).

The class I MHC molecule is a ternary complex consisting of a polymorphic heavy chain, a noncovalently associated light chain beta 2-microglobulin, and a small peptide (8-10 residues) (3-8). The peptide binding cleft is formed by the alpha 1 and alpha 2 domains of the heavy chain. For effective CD8+ T cell responses, class I MHC molecules must bind many peptides of diverse sequence in sufficient abundance for long periods of time. Typical half-lives of immunodominant peptides are greater than 20 h at 37 °C (9, 10). Peptides bind to class I MHC primarily through the invariant peptidic termini (11-13). In addition, the polymorphic residues within the peptide binding groove create specificity pockets that select specific amino acids in the peptide (14, 15). The specificity pockets for HLA-A2.1 (A2) are found close to the peptidic termini and are complementary in shape and charge to residues 2 (P2) and the last residue of the peptide (POmega ). The specificity pockets play a large role in binding affinity to A2 (16, 17) but not to all class I MHC (18). The result of this set of interactions in A2 is the binding of the ends of the peptide, leaving the center relatively free of interactions. The center of the peptide bulges out of the peptide binding cleft, and the main chain rarely traverses the same path in two different peptides (19). The co-crystal structures of class I MHC and TCR show that the means of engagement between pMHC and TCR are conserved (20-24). The TCR binds in a diagonal manner with the TCR alpha  chain interacting with the carboxyl end of the MHC alpha 2 helix and the TCR beta  chain interacting with the carboxyl end of the MHC alpha 1 helix. The CDR3 regions of the TCR alpha  and beta  chains interact with the center of the peptide (P5-P7 depending on the peptide) (25, 26).

Two aspects about peptide binding to class I MHC are explored by the experiments presented here. The first aspect is an assumption that each amino acid in the peptide binds independently of one another to enhance or detract from the overall binding affinity. Using this assumption, a popular algorithm was designed to predict peptide epitopes that bind well. This algorithm is predicated on the assumption that each residue binds independently (27). Although this algorithm predicts many good binding peptides from proteins of interest (28-32), for unknown reasons it fails to predict accurately the results of single amino acid substitutions. The second aspect of peptide binding explored here is that residues at the anchor positions do not affect the conformation of residues elsewhere in the peptide. If one assumes that each residue binds independently, homologous substitutions would be the best choice to amplify and activate T cells specific for the parental antigen (33-35). The clear choice for modification is the anchor residues because they point into the binding cleft and are restricted in space by the specificity pockets. Thus, the conformation flexibility of the peptide backbone should be limited, and any alterations in the structure caused by the anchor substitution would be expected to be local and small. This idea has been used to design peptides with increased affinity for class I MHC to enhance CTL stimulation. This approach has been successful in some cases (36-38) and varied in others (39). T cells stimulated using altered-peptide ligands (APL) are not necessarily the same population of T cells (40). This change in reactivity may be a result of interactions between the single amino acid changes and the MHC or between the substitutions and the other amino acids within the peptide. Our studies provide an explanation for the instances in which alteration reduces or eliminates reactivity. These data show that substitutions at the anchor positions can directly alter the conformation of the residues at the center of the peptide and conversely that substitutions in the center can cause large changes at the termini.

We examined binding of a selection of known immunologically recognized peptide ligands from the tyrosine kinase family member HER-2/neu to the class I MHC molecule A2. HER-2/neu is overexpressed in ~30% of patients with breast cancer and similarly in all adenocarcinomas examined. Despite the presence of CTL that recognize these peptides bound to A2, the tumors are not eliminated. These HER-2/neu-derived peptides contain appropriate anchor residues but still bind poorly to A2 molecules (41). One proposed explanation for inefficient tumor killing is that the peptide antigens bind poorly to A2, and these complexes are not stable enough to be recognized well by HER-2/neu peptide-specific CTL. Our long term goal is to design high affinity APL for cancer immunotherapy. We have examined binding of one of these poor-binding peptides, GP2 (IISAVVGIL), to design a ligand for immunotherapy. The crystallographic structure of GP2 co-crystallized with A2 (A2GP2) shows that the center of the peptide is disordered and apparently does not make stabilizing contacts with the peptide binding cleft (41). GP2 has anchor residues that are present in high affinity peptides (isoleucine at position 2 and leucine at position 9) (42). Substitution of these anchor residues with amino acids most preferred by A2 increased the binding affinity, but not significantly (41).

Based on the crystallographic structure of GP2 bound to A2, we designed a new APL in which we substituted the position 5 (P5) valine with leucine (V5L). We hypothesized that the larger leucine would fit into a hydrophobic pocket in the peptide binding cleft under the alpha 2 alpha  helix where the smaller valine could not reach. We synthesized a series of peptides that included the V5L substitution in combination with our anchor substitutions to maximize binding affinity. Measurements of binding affinity and peptide off-rates showed that the enhancement in binding of double-substitution peptides was not the sum of the increases from the single-substitution peptides. We interpreted this to mean that there are interactions between residues in the peptide or changes in the peptide structure. To understand these interactions, the crystallographic structures of A2 bound to the GP2 variants I2L/V5L (A2I2L/V5L) and I2L/V5L/L9V (A2I2L/V5L/L9V) were determined. These structures show that the peptide residues interact and that the TCR contact residues alter their positions depending on the identity of the anchor residue. Therefore, homologous substitutions anywhere in the peptide may have large unintended consequences in T cell recognition.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Peptides-- The peptides used in this study are listed in Table I. All peptides were synthesized by the Peptide Synthesis Facility at the University of North Carolina, Chapel Hill. The peptides were purified to greater than 95% purity as confirmed by reversed phased chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Peptides were dissolved in 100% dimethyl sulfoxide, 10 mg/ml by weight. Final concentrations were determined by amino acid analysis by the Protein Chemistry Laboratory in the Department of Chemistry, University of North Carolina, Chapel Hill.

                              
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Table I
Binding data of GP2 variant peptides to A2
Residues substituted with respect to wild type peptide are shown in boldface. Tm is the temperature (°C) at which 50% of protein is denatured as measured by circular dichroism. Kr is the relative binding constant as determined by the T2 cell surface assembly assay. t1/2 is the half-life of A2-peptide complexes (in hours) as determined by the T2 cell surface stability assay. The error in the Tm is the sum of machine and curve fit errors and is typically about 0.5 °C.

Preparation of HLA-A2.1-Peptide Complexes-- HLA-A2.1-peptide complexes were prepared as described previously (43). Briefly, residues 1-275 of A2 and residues 1-99 of beta 2-microglobulin were produced in Escherichia coli as inclusion bodies. Peptide, solubilized beta 2-microglobulin, and A2 heavy chain, solubilized in 8 M urea, were rapidly diluted into folding buffer (100 mM Tris, pH 8.0, 400 mM L-Arg, 10 mM GSH, 1 mM GSSG, and protease inhibitors) at molar ratios of 10:2:1, respectively. The solution was incubated at 10 °C for 36-48 h, concentrated by ultrafiltration (Amicon), and purified by high pressure liquid chromatography gel filtration (Phenomenex, BioSep-SEC-S2000).

Thermal Denaturation Studies-- The thermal denaturation properties of A2-peptide complexes were determined as described previously (12). Purified A2-peptide complexes were exchanged into a 10 mM KH2/K2HPO4 buffer, pH 7.5, and adjusted to final concentration of 4-12 µM. Thermal denaturation curves (melting curve) of MHC-peptide complexes were recorded by monitoring the change in circular dichroic (CD) signal at 218 nm as a function of temperature from 4 °C to 95 °C on an AVIV 62-DS spectropolarimeter (Aviv Associates, Lakewood, NJ). The final melting curves were the average of at least three measurements for each complex. Tm values were calculated as the temperature at which 50% of the complexes are unfolded.

Cell Surface Stabilization Assays-- The ability of peptide to stabilize A2 on the surface of T2 (ATCC CRL-1992) cells was determined as described previously (10). Briefly, T2 cells (2.5 × 105 cells/well) were incubated overnight in AIM V medium (Life Technologies, Inc.) with varying concentrations of peptide. The following morning, cells were stained with the A2-specific monoclonal antibody BB7.2. After two washes with wash buffer (1 × phosphate-buffered saline, 2% fetal bovine serum, 0.1% sodium azide), the cells were incubated for 30 min at 4 °C with a 1:50 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody (Southern Biotechnologies). Fluorescence was detected on a FACScan (Becton-Dickinson, Lincoln Park, NJ). The data were then normalized to the mean channel fluorescence for the index peptide ML at 50 µM. The ML peptide (MLLSVPLLL) is derived from the signal sequence of calreticulin and has a hydrophobicity similar to that of the peptides used in this study.

Cell Surface Half-life Assay-- The half-life of A2-peptide complexes on the surface of T2 cells was determined as described previously (10). Briefly, T2 cells (8 × 105 cells/well) were incubated overnight in AIM V medium with 50 µM peptide. Cells were incubated in RPMI-1650, 10% fetal calf serum, 10 µg/ml brefeldin A for 1 h. Because this concentration of brefeldin A is toxic to the cells if they are exposed for long periods of time, the cells were transferred and maintained at 0.5 µg/ml through FACScan analysis. Cells were then stained with BB7.2 at various time points and analyzed by flow cytometry as described above. The mean fluorescence for the peptide at each time point minus the mean fluorescence of T2 cells incubated without exogenous peptide was calculated and normalized to the maximum level of fluorescence for each APL (at t = 0).

Crystallization, Data Collection, and Data Processing-- Crystals were grown by the hanging drop vapor diffusion method. Crystals were grown from 14-20% polyethylene glycol 8000, 25 mM MES, pH 6.5, for both A2I2L/V5L/L9V and A2I2L/V5L over the course of 2 days by microseeding. Crystallographic data for both structures were collected at 100 K in house (University of North Carolina Macromolecular Crystallography Facility) on a RIGAKU RU200 equipped with RAXIS IIC imaging plate detector and Oxford Cryostream. Data were collected from a single crystal of A2I2L/V5L/L9V and from two crystals of A2I2L/V5L. Data for both structures were integrated with DENZO and intensities scaled with SCALEPACK (44). The statistics for each data set for both structures are given in Table II.

                              
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Table II
Crystallographic and refinement statistics

Structure Determination and Refinement-- Both structures were determined by molecular replacement using AMoRe (45) within the CCP4 program suite (46). The crystals are triclinic, space group P1 with two molecules per asymmetric unit.The A2-hepatitis peptide complex (PDB accession code 1HHH) was used as the search model (47). The search model was divided into three pieces: the peptide binding superdomain (alpha 1alpha 2), the alpha 3 domain, and the beta 2-microglobulin light chain. Rigid body refinement was performed in CNS (48-50) leaving the alpha 1alpha 2, alpha 3, and beta 2-microglobulin as three separate rigid bodies. Nine rounds of torsional dynamics refinement with CNS and manual intervention with O (51) were performed. NCS restraints for regions not involved in crystal contacts were maintained, and a model was built for one copy in the asymmetric unit and the second generated using the NCS operators. To reduce model bias, peptide was not included in the initial three rounds of refinement. The electron density maps were generated using DM (46) using the functions for 2-fold noncrystallographic averaging, histogram matching and solvent flattening. 144 water molecules for A2I2L/V5L/L9V and 35 waters for A2I2L/V5L were added to the structure using the program ARP (52) combined with REFMAC and confirmed by visual inspection of the electron density maps. Refinement statistics for each model are given in Table II.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stabilization Derived from Individual Anchor Substitutions Is Not Additive in Double-substitution Peptides-- Previous studies had shown that substitutions at anchor positions increased thermal stability of A2GP2 (41). The thermal stability of the peptides given in Table I shows that the individual anchor substitutions generate increases in Tm from 2.4 °C (A2L9V) to 5.8 °C (A2I2L). If stabilization of binding were the sum of the increases, the double substitution should generate 8.4 °C increased thermal stability, but the observed difference is 6.1 °C (A2I2L/L9V), suggesting that the mechanism of stabilization is not entirely peptide position-independent. The T2 assays of binding and kinetics of dissociation confirm that there is an improvement in the double-anchor substitution peptide. The binding is better (smaller Kr), and dissociation is slower (longer t/2) for the double anchor variant, but the expected changes in the values cannot be examined for the Kr because we cannot measure the value for A2L9V.

A Substitution at the Center of the Peptide Designed to Improve Binding Affinity Actually Reduces Binding Affinity by Itself and Interacts with Anchor Substitutions Unexpectedly-- Based on the crystallographic structure of GP2 bound to A2 (41), we designed a new peptide in hopes that it would bind with higher affinity and reduce the disorder in the center of the peptide. In the structure, a large hydrophobic pocket was found near the P5 valine under the alpha 2 alpha  helix. We hypothesized that a leucine in place of the valine at position 5 in the center of peptide would be more complementary to the size and shape of the pocket. The V5L peptide was synthesized and tested for binding affinity using T2 and CD assays. As can be seen in Table I, the V5L peptide actually binds worse than does GP2. In fact, it binds so poorly that A2V5L cannot be isolated in sufficient quantity to use in CD or structural studies. Combinations of anchor substitutions and the V5L substitutions were synthesized to determine whether better anchors would facilitate use of this hydrophobic pocket by the leucine at P5. It quickly became clear that these substituted residues were interacting in unexpected ways. For example, the Tm of A2L9V is 38.8 °C, and the Tm of A2V5L/L9V is also 38.8 °C, suggesting that the V5L substitution makes no difference to the stability of the complex. However, as described above, V5L alone binds with lower affinity compared with GP2, suggesting that it detracts from binding affinity. Confirming this, the Tm for the A2I2L/V5L double substitution is 39.0 °C, which is 3.2 °C lower than the single substitution I2L. Similarly, the Tm for the triple substitution A2I2L/V5L/L9V is 39.5 °C, which is 3.0 °C lower than the double substitution A2I2L/L9V. These unexpected interactions as measured by CD are substantiated by the binding assays using T2 cells (Fig. 1 and Table I). To understand these interactions, we decided to determine the crystal structures of some of these complexes.


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Fig. 1.   Thermal denaturation as measured by circular dichroism shows unpredicted interactions between residues in the peptide. Panel A, thermal denaturation curves of complexes of A2 bound to GP2 variants. Each curve is the average of three independent experiments using 4-12 µM protein. The Tm is the temperature at which 50% of the complexes are unfolded. The error associated with this type of measurement is the sum of the error in the temperature controller and the curve fitting error. This is ~0.5 °C for each complex. Panel B, cell surface A2 was stabilized on T2 cells by the addition of the indicated amounts of peptide. The amount of A2-peptide on the cell surface was measured by flow cytometry using the A2-specific monoclonal antibody, BB7.2. Panel C, the rate of loss of cell surface A2-peptide complexes was measured by treating the peptide-pulsed cells (as in panel B) with brefeldin A to halt vesicular transport. Aliquots of cells were removed at the indicated times and the remaining A2-peptide on the cells determined by incubating with BB7.2. Error bars are the S.E.

The Crystallographic Structures of A2I2L/V5L/L9V and A2I2L/V5L Confirm That the Individual Residues Interact-- The molecular replacement solutions for the two complexes were unambiguous and gave correlation coefficients of 79% for A2I2L/V5L/L9V and 75% for A2I2L/V5L using 1HHH as a search model (47). These initial models were refined in CNS (48-50), and peptide was omitted during the first three stages of refinement (until the Rwork was below 30%) in both structures to reduce model bias. Density modification was performed with DM (46) to generate unbiased averaged electron density maps. The final structures are well defined in the electron density maps with average real space correlation coefficients of 78.4 and 80.7% with all fragments of A2I2L/V5L and A2I2L/V5L/L9V. The final models have an overall Rfree of 29.0% from 50 to 2.3 Å for A2I2L/V5L and 28.6% from 50 to 2.25 Å for A2I2L/V5L/L9V with good stereochemistry and no residues in the disallowed regions of a Ramachandran plot (Table II).

In both structures, the positions of the peptidic termini are unambiguous. However, the electron density at the center of the peptide is not well defined in both I2L/V5L and I2L/V5L/L9V peptides as was seen in the GP2 peptide (41). The peptides in the two molecules in the asymmetric unit are not equivalent in these structures, demonstrating some of the dynamics that are known to be in the system. These differences in the peptides are not caused by crystal contacts.

A2I2L/V5L Structure-- The electron density for the I2L/V5L peptide in molecule 2 (MOL2) in the asymmetric unit is broken at valine at P6 and glycine at P7 (Fig. 2B). This is similar to what was observed in the GP2 peptide in the A2GP2 crystal structure (41). However, in molecule 1 (MOL1) the peptide density is continuous over the main chain of the peptide (Fig. 2A). However, the side chain for residue 6 is undefined, and the temperature factors are higher for the central residues in the peptide, demonstrating that the central residues in molecule 1 have greater disorder than the termini. The orientation of side chain of leucine at P5 is different between the two NCS symmetry-related molecules of A2I2L/V5L (a rotation of ~83o of the Calpha -Cbeta bond), and they refine to these different positions regardless of the starting position before CNS refinement. Although there is not sufficient density to have absolute confidence in the position of the valine at position 6, there is sufficient electron density to strongly suggest that they orient in opposite directions. It appears that the P6 valine in molecule 2 points diagonally toward the alpha 2 alpha  helix and down into the cleft, and the P6 valine in molecule 1 points toward the alpha 1 alpha  helix.


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Fig. 2.   Averaged omit electron density maps contoured at 1sigma show that the central residues of I2L/V5L and I2L/V5L/L9V are disordered as in the structure of A2GP2. Panel A, averaged omit map of peptide GP2I2L/V5L in molecule 1 (MOL1) in the asymmetric unit. Panel B, averaged omit map of peptide molecule 2 (MOL2) in the GP2I2L/V5L asymmetric unit. Panel C, averaged omit map of peptide GP2I2L/V5L/L9V in molecule 1 of asymmetric unit. Panel D, averaged omit map of peptide GP2I2L/V5L/L9V in molecule 2 of asymmetric unit. All molecules displayed have A2 removed for clarity, and the maps are contoured at 1sigma with a cover radius of 1 Å.

A2I2L/V5L/L9V Structure-- Although there are differences in the electron density for the peptides in molecules 1 and 2 in the asymmetric unit of the A2I2L/V5L/L9V, the differences are much smaller than those observed for the two copies of A2I2LV5L and do not affect the orientations of the peptide in either model. There is a break in the electron density at valine at P6 and glycine at P7 in both symmetry-related molecules as in the A2GP2 structure, and the orientation of valine at P6 cannot be interpreted in either molecule (Fig. 2, C and D). The electron density for the leucine at P5 is not well defined, but the direction of the electron density clearly indicates the orientation of the side chain is toward solvent in both copies in the asymmetric unit.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

For an effective immune response, it is necessary that class I MHC present antigenic peptides for long periods of time on the cell surface to allow detection of these complexes by circulating T cells (53). Many tumor cells appear to escape the immune response because antigenic peptides do not bind well to the class I MHC molecule that present them (54, 55). If peptide does not bind efficiently to the MHC molecule, circulating T cells will not recognize the pMHC complex, and cells presenting them will not be eliminated. To enhance the binding affinity of antigenic peptides to class I MHC molecules, it is necessary to understand the forces that determine the binding affinity of peptide to class I MHC. We have examined a poor-binding peptide antigen derived from the tyrosine kinase family member HER-2/neu.

Our initial studies were focused on improving the binding affinity of GP2 to A2 by producing variants of GP2 at the anchors. The long term goal is to use the variant peptide of GP2 to stimulate a vigorous GP2-specific CTL response in the cancer patient or as a vaccine in a healthy person. Because the prevalent belief is that each amino acid acts independently to generate positive or negative effects to binding energy (27), we expected to be able to generate the best binder in a stepwise fashion; that is, we saw an increase in thermal stability with I2L and L9V peptides bound to A2, and we expected to be able to add them together to get the best binding peptide (I2L/L9V). The data showed that the increased affinity generated by this approach was not as great as we expected.

We determined the crystal structure of A2GP2 and during our analysis of the structure, and we hypothesized that if the valine at P5 was a leucine, it could take advantage of a hydrophobic pocket under the alpha 2 alpha  helix. However, our data clearly show that this substitution also did not improve binding affinity. More surprisingly, we saw that combinations of anchor substitutions with the V5L substitution resulted in unexpected changes to the thermal stability of the complex. In one case, the substitution had no effect (V5L/L9V), and in others, it made a large difference (I2L/V5L and I2L/V5L/L9V peptides). To understand this phenomenon, we determined the crystal structures of A2I2L/V5L and A2I2L/V5L/L9V.

The crystallographic structures show that the electron density at the centers of the I2L/V5L and I2L/V5L/L9V peptides is disordered in most cases (three out of four copies), highly mobile, and poorly fit into the electron density in the last case (A2I2L/V5L molecule 1, Fig. 2A, and Table III). These data show that the substitutions at the anchors and at P5 have not decreased the flexibility at the center of the peptide, and hence the binding affinity did not improve substantially.

                              
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Table III
Comparison of main chain temperature factors (B in Å2) and real space correlation coefficient (RSCC) between peptides GP2, I2L/V5L, and I2L/V5L/L9V in the two molecules in the asymmetric unit
The GP2 peptides are identical for both molecules in the asymmetric unit.

Based on the crystal structure of A2GP2, we expected the substituted leucine at the P5 to point down into the peptide binding cleft under the alpha 2 alpha  helix and to fit into a hydrophobic pocket there. The closest we came to predicting the orientation was in the structure of A2I2L/V5L. A comparison of the structure of GP2 bound to A2 with the structure of A2I2L/V5L shows that the leucine points in the same direction as valine in molecule 1, but the side chain points away from the hydrophobic pocket (Fig. 3A). In molecule 2 of A2I2L/V5L, the leucine is more solvent-exposed and is nowhere close to the pocket (Fig. 3B). One of the surprises found in this analysis was that there were many changes seen in the positions of the amino acids past the P5 residue. Although there is not a great deal of confidence in the absolute positions of these central residues, it is clear that the density defines very different paths for the peptide molecule 1 (Fig. 3A). The effect of this substitution on the carboxyl terminus in molecule 2 is even clearer. The position of the P5 residue alters the position of all the residues from P6 to P9 in molecule 2 such that the carboxylate is displaced by 1.0 Å (Fig. 3B). The error associated with this model is 0.31 Å. Interestingly, an examination of the termini of these peptides (except for one copy of I2L/V5L/L9V) shows high temperature factors for the carboxyl termini compared with the amino terminus (Table III). This may reflect the more buried nature of the P1 residue compared with the P9 residue.


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Fig. 3.   Substitutions of amino acids in the peptide alter the position of other residues in the peptide. Panel A, superpositioning the alpha 1alpha 2 peptide binding superdomains of A2GP2 and A2I2L/V5L molecule 1 (MOL1) shows that the central residues are in very different positions as a result of the double substitution. Panel B, superpositioning A2GP2 and molecule 2 (MOL2) of A2I2L/V5L demonstrates the changes that occur from positions 6-9 in the peptide as a result of the changes. Panel C, comparisons of GP2 and I2L/V5L/L9V show moderate changes, and most are focused on the V5L substitution. Panel D, a similar comparison with molecule 2 in I2L/V5L/L9V shows even fewer changes. Panel E, a comparison of the peptides in I2L/V5L and I2L/V5L/L9V (both molecule 1 (MOL1)) shows alterations at the carboxyl end of the peptide as a result of the change L9V. Panel F, a similar comparison (with molecule 2) illustrates, as did panel C, that position 5 is altered drastically as a consequence of the L9V substitution. The figure was generated with GRASP (60).

The most apparent difference between the structures of GP2 and I2L/V5L/L9V peptides is the position of the leucine side chain at P5. In A2I2L/V5L/L9V, the leucine points toward solvent away from A2 and does not interact with any residue of the peptide binding superdomain regardless of which molecule is examined (Fig. 3, C and D).

A comparison of the structures of A2I2L/V5L and A2I2L/V5L/L9V illustrates the types of differences that may occur when changing anchor residues (Fig. 3, E and F, comparing both molecules 1 or molecules 2). In these peptides, the only difference is a substitution of the GP2 P9 leucine with valine. The result of the substitution can be a 90° rotation of the Calpha -Cbeta bond at P5 as observed in molecule 1 (Fig. 3E). The rotation moves the leucine from pointing toward the alpha 1 alpha  helix (A2I2L/V5L) to pointing toward solvent (A2I2L/V5L/L9V). The P5 residue is directly in the center of the peptide, and based on the co-crystal structures of pMHC and TCR would directly contact the CDR3 regions of the alpha  and beta  chains of the TCR. Or the result of this substitution can be a reordering of the positions of all of the atoms nearby at positions 7-9 as observed in molecule 2 (Fig. 3F).

These data show that large structural changes may occur by small homologous substitutions in the center or in the anchors of the peptide. These structural changes can greatly change TCR recognition. The observed changes in A2I2L/V5L/L9V and A2V5L/L9V are significantly larger than those seen previously to change TCR reactivity (22, 56). In particular, changes as small as a substitution of tyrosine for phenylalanine at P1 can cause only localized changes about the P1 residue, but significant changes in T cell recognition (56). These changes are much more dramatic. Immunization with I2L/L9V peptide generates small but reproducible CTL reactivity in A2Kb transgenic mice. Similarly, we are able to generate small responses to I2L/V5L/L9V, but we are unable to generate any responses to the I2L/V5L or V5L/L9V peptides (data not shown). These CTL data are not significant in themselves, but they confirm the not unexpected idea that TCR recognition is different with respect to these peptides.

In summary, the data presented here show that substitutions in the center of a peptide bound to class I MHC may affect the positions of all of the residues within the peptide. In addition, small homologous substitution in the anchor residues can dramatically alter the TCR-contacting residues. Clearly, the presence of substituted residues may alter the positions of nearly all of the other residues even when the substitution is a minor homologous substitution as is seen in the case of peptides I2L/V5L and I2L/V5L/L9V. These types of changes were implicated previously in a study of H-2Kb with a panel of antibodies (57) which showed unexpected changes in antibody reactivity with a series of peptide substitutions. Similarly, homologous amino acid changes in TCR-contacting residues show greatly changed reactivity (58). This may be explained in terms of a change in the affinity between the MHC-peptide complex and the TCR but could also be the result of secondary changes in the peptide conformation as observed here. Examinations of clones induced by immunization of anchor-substituted APL in a clinical trial showed large differences in the set of T cells expanded compared with immunization with tumor-infiltrating lymphocytes (59). These data are surprising because substitutions have been shown to have very limited effects on the path of the peptide (56).2 One difference between those studies and these data is the relative binding affinity of the peptides studied. GP2 is a poor-binding peptide, and the others bind much better. Perhaps in the case of weak affinity, substitutions have a more dramatic effect. This may have important implications in cancer immunotherapy because most peptides studied are of low affinity (41).

    ACKNOWLEDGEMENTS

We thank Carrie Barnes for excellent technical assistance, members of the Collins and Frelinger laboratories for stimulating discussions, and Dr. Jeffrey Frelinger for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by Department of Defense Grant DAMD17-97-1-7052.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1EEYI2L/V5L/L9V (A2) and 1EEZ (A2I2L/V5L)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org).

|| To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of North Carolina, CB 7290, 804 M. E. Jones Bldg., Chapel Hill, NC 27599. Tel.: 919-966-6869; Fax: 919-962-8103; E-mail: edward_collins@med.unc.edu.

Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M010791200

2 A. K. Sharma, J. J. Kuhns, S. Yan, R. H. Friedline, B. Long, R. Tisch, and E. J. Collins, unpublished data.

    ABBREVIATIONS

The abbreviations used are: MHC, major histocompatibility complex; TCR, T cell receptor(s); CTL, cytotoxic T lymphocyte(s); pMHC, peptide-MHC complex; A2, HLA-A2.1; P2 and P5, peptide specificity pocket for residue 2 and 5, respectively; POmega , A2 specificity pocket for last residue of peptide; APL, altered-peptide ligands; A2GP2, GP2 co-crystallized with A2; A2I2L/V5L, A2 bound to GP2 variants I2L/V5L; A2I2L/V5L/L9V, A2 bound to GP2 variants I2L/V5L/L9V; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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