From the Departments of 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
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ABSTRACT |
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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.
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 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 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.
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 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.
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
( 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 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
C 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.
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 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2-microglobulin, and a small peptide (8-10 residues)
(3-8). The peptide binding cleft is formed by the
1 and
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 (P
). 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
chain interacting with the carboxyl end of the MHC
2 helix and the
TCR
chain interacting with the carboxyl end of the MHC
1 helix.
The CDR3 regions of the TCR
and
chains interact with the center
of the peptide (P5-P7 depending on the peptide) (25, 26).
2
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
Binding data of GP2 variant peptides to A2
2-microglobulin were
produced in Escherichia coli as inclusion bodies. Peptide,
solubilized
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).
Crystallographic and refinement statistics
1
2), the
3 domain, and the
2-microglobulin
light chain. Rigid body refinement was performed in CNS (48-50)
leaving the
1
2,
3, and
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
2
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.
-C
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
2
helix and down into the cleft, and the P6 valine in molecule 1 points toward the
1
helix.
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[in a new window]
Fig. 2.
Averaged omit electron density maps contoured
at 1 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 1
with a cover radius of 1 Å.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2
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.
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
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 2
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.
|
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 C-C
bond at P5 as observed in molecule 1 (Fig.
3E). The rotation moves the leucine from pointing toward the
1
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
and
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).
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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.
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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.
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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;
P, 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.
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