X-ray diffraction studies as well as
structure-activity relationships indicate that the central part of
class I major histocompatibility complex (MHC)-binding nonapeptides
represents the main interaction site for a T cell receptor. In order to
rationally manipulate T cell epitopes, three nonpeptidic spacers have
been designed from the x-ray structure of a MHC-peptide complex and
substituted for the T cell receptor-binding part of several antigenic
peptides. The binding of the modified epitopes to the human leukocyte
antigen-B*2705 protein was studied by an in vitro
stabilization assay, and the thermal stability of all complexes was
examined by circular dichroism spectroscopy. Depending on their
chemical nature and length, the introduced spacers may be classified
into two categories. Monofunctional spacers (11-amino undecanoate,
(R)-3-hydroxybutyrate trimer) simply link two anchoring
peptide positions (P3 and P9) but loosely contact the MHC binding
groove and thus decrease more or less the affinity of the altered
epitopes to human leukocyte antigen-B*2705. A bifunctional spacer
((R)-3-hydroxybutyrate tetramer) not only bridges the two distant anchoring amino acids but also strongly interacts with the
binding cleft and leads to a 5-fold increase in binding to the MHC
protein. To our knowledge, this is the first report of a nonpeptidic
modification of T-cell receptor binding residues that significantly
enhances the binding of altered peptide ligands to their host MHC
protein. The presented modified ligands constitute interesting tools
for perturbing the T cell response to the parent antigenic peptide.
 |
INTRODUCTION |
Class I MHC1
molecules are highly polymorphic proteins that play a key role in
immune surveillance by presenting foreign peptides to cytotoxic T
lymphocytes (1). The molecular mechanisms of peptide selection have
been characterized by x-ray diffraction studies of several MHC proteins
in complex with either a peptide pool or single ligands (2). Peptides,
generally nonamers, tightly bind to conserved MHC residues in a
sequence-independent manner at their N and C termini (3), whereas the
central part of the bound peptide bulges out of the binding groove (4).
Peptide specificity is governed by the position and chemical nature of some anchoring side chains (often P2, P3, and P9) that bind to MHC
polymorphic pockets (5, 6). Complementary to x-ray structure determinations, sequencing self-peptides naturally bound to MHC proteins allows the determination of peptide binding motifs (7, 8) and
thus the identification of conserved amino acids responsible for MHC
binding (named dominant anchors, generally at positions P2 and P9) and
more variable residues hypothesized to account for TcR recognition
(usually in the central part of the peptide sequence, from P4 to P8).
Peptide mutation (9, 10) as well as recently determined x-ray
structures of 
TcRs in complex with a MHC-peptide (11, 12)
unambiguously support this assumption. Since some class I MHC
alleles are associated with either susceptibility or resistance
to human diseases (13-15), altering TcR contact residues of T cell
epitopes has been proposed for designing altered peptide ligands with
TcR antagonist properties (16), leading to in vivo T cell
anergy (17). However, natural peptides cannot be easily used as
immunosuppressors because of poor enzymatic stability and
pharmacokinetic properties (18). Herewith, we describe the substitution
of nonpeptidic moieties for the TcR contact amino acids of several T
cell epitopes naturally presented by the class I MHC protein B*2705,
which is strongly linked to severe inflammatory diseases like
ankylosing spondylitis (13) or reactive arthritis (19). Some reports in
which a similar strategy has been followed (20-22) show that the
altered peptide ligands still form stable complexes with their host MHC
protein but often present a reduced affinity relative to the parent
peptide. The present study describes a novel oligomeric spacer able not
only to link two MHC anchoring positions (P3 and P9) but also to
significantly improve binding to the restricting class I MHC
protein.
 |
EXPERIMENTAL PROCEDURES |
Computer-assisted Ligand Design--
Molecular mechanics and
dynamics calculations were carried out using the AMBER 4.1 package (23), using the parm94 parameter set (24) and an all-atom force
field representation. Force field parameters for the ester group were
taken from the literature (25). Atomic charges for the Aua and HB
monomers were calculated using the GAUSSIAN 94 package (26)
and the HF/6-31G* basis set by fitting atom-centered charges to an
ab initio electrostatic potential, using the RESP method
(27) according to a previously described procedure (28). Atomic charges
for both new monomers are listed in Table
I.
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Table I
Restrained electrostatic potential-derived point charges, calculated
using the RESP method (26) from GAUSSIAN 94 HF/6-31G* electrostatic
potentials
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Initial coordinates for the MHC-ligand complexes were obtained from the
x-ray structure of HLA-B*2705 (3) as described previously (20, 29). The
spacers were substituted for the natural pentapeptide sequence using
the SYBYL modeling package (TRIPOS Association, Inc., St. Louis, MO).
From a starting fully extended conformation, dihedral angles of the
main chain between P3 and P9 were modified by hand in order to
reproduce a correct trans geometry for the newly introduced amide or
ester bonds. The ligand was first relaxed by 500 steps of conjugate
gradient energy minimization while maintaining the protein fixed. It
was then submitted to a 100-ps simulated annealing protocol in order to
sample the broadest conformational space accessible. Starting with
random velocities assigned at a temperature of 1000 K, the ligand was
first coupled for 50 ps to a heat bath at 1000 K using a relatively
weak temperature coupling constant
(0.2 ps) and then linearly
cooled down to 50 K for the next 50 ps while
was strengthened to a
value of 0.05 ps. During these 100 ps, no protein atoms were allowed to
move. The last conformer was then solvated in a 10-Å-thick TIP3P water
shell. Energy minimization of the ligand, of the MHC-ligand complex,
followed by 200-ps molecular dynamics simulation of the fully solvated
MHC-ligand pair was performed as previously reported (20).
Synthesis of the Modified Peptides--
Ligands 1-8 (Table
II) were obtained by automated
solid-phase peptide synthesis using a Fmoc/tert-butyl
protecting strategy. Chain elongation was performed by a robot system
(Syro Multi-Syn-Tech, Bochum, Germany) with a subsequent manual
deprotection and analysis. Fmoc-protected amino acids were coupled to
the diisopropylcarbodiimide-activated carboxyl terminus in 10-fold
excess using 1-hydroxybenzotriazole as a coupling reagent. The final
peptide was simultaneously cleaved from the resin and deprotected by
the addition of trifluoroacetic acid with thiocresole and thioanisole
as scavengers. The peptides were precipitated and washed with ice-cold
ether and further lyophilized from water. Natural as well as nonnatural
peptides were analyzed by reverse phase high performance liquid
chromatography (Merck-Hitachi, Darmstadt, Germany) on a nucleosil 5µ,
C-18 column (125 × 3 mm) at a flow rate of 600 µl/min.
Absorbance was measured at 220 nm. The solvent system consisted of
0.1% trifluoroacetic acid in water (buffer A) and 0.1%
trifluoroacetic acid in acetonitrile (buffer B). A linear gradient from
10 to 60% B in 30 min was applied. Furthermore, peptides were analyzed
by ion spray mass spectrometry on a triple quadrupole mass
spectrometer, APII III, with a mass range of m/z
10-2400 equipped with an ion spray interface (Sciex, Thornhill,
Canada). The mass spectrometer was operated in positive ion mode under
conditions of unit mass resolution for all determinations.
The synthesis of ligands 9-12 will be reported
elsewhere.2
Epitope Stabilization Assay--
The quantitative assay used was
described previously (30). Briefly, RMA-S transfectants expressing
B*2705 or B*2704 were used. These are murine cells with impaired
TAP-mediated peptide transport and low surface expression of (empty)
class I MHC molecules, which can be induced at 26 °C (31) and
stabilized at the cell surface through binding of exogenously added
ligands. These cells were incubated at 26 °C for 24 h. After
this, they were incubated 1 h at 26 °C with 10
4
to 10
9 M peptides, transferred to 37 °C,
and collected for flow microcytometry analysis with the ME1 monoclonal
antibody (IgG1, specific for HLA-B27, -B7, and -B22) (32) after 4 h for B*2705 or after 2 h for B*2704. The determinant recognized
by ME1 is not affected by bound peptides or by polymorphism in these
two subtypes (data not shown). Binding of a given ligand was measured
as its C50. This is its molar concentration at 50% of the
fluorescence obtained with that ligand at 10
4
M. Ligands with C50
5 µM were
considered to bind with high affinity, since these were the values
obtained for most of the natural B27-bound peptides. C50
values between 5 and 50 µM were considered to reflect
intermediate affinity. C50
50 µM indicated low affinity. Binding of peptide analogs was measured as the
concentration of the peptide analog required to obtain the fluorescence
value at the C50 of the unchanged peptide. This was
designated as EC50. Relative binding was the ratio between
the EC50 of the peptide analog and the C50 of
the corresponding unchanged peptide.
HLA-B*2705 Expression and Purification--
A cDNA encoding
for human
2-microglobulin (gift of Dr. C. Vilches,
Clinica Puerta de Hierro, Madrid) was cloned into a pGex vector
(Amersham Pharmacia Biotech), yielding a fusion protein with
glutathione S-transferase. Escherichia coli cells
transformed with this pGex vector were grown under vigorous shaking in
LB broth for 24 h at 25 °C after induction with
isopropyl-1-thio-
-D-galactopyranoside. Cells were frozen
at
70 °C, thawed, suspended in TBS (20 mM Tris, 150 mM NaCl, pH 8.0), and lysed by the addition of lysozyme and brief sonication. The crude extract was passed over a
glutathione-agarose column (Sigma), and after extensive washing with
TBS, the
2-microglobulin was eluted by thrombin cleavage
as a single band at 11 kDa (SDS-polyacrylamide gel
electrophoresis).
The HLA-B*2705 heavy chain was affinity-purified under denaturing
conditions as a His6 fusion protein. The expression vector was obtained by subcloning the cDNA encoding for the first
extracellular 274 amino acids (gift of Dr. K. C. Parker, National
Institutes of Health, Bethesda) into the polycloning site of the
oligohistidine vector pQE30 (Quiagen) with the restriction
endonucleases BamHI and HindIII. The heavy chain
was expressed in E. coli at 35 °C for 2 h after
induction with isopropyl-1-thio-
-D-galactopyranoside. Longer expression times led to an increase of immature or degraded heavy chains. Inclusion bodies were prepared using a standard procedure
(33) and solubilized in 8 M urea, 20 mM Tris,
150 mM NaCl at pH 8.0. Purification on a
nickel-nitriloacetate-agarose column led to the HLA-B*2705 heavy chain
with two minor impurities of lower molecular weights consisting of
truncated heavy chains.
Folding of the MHC Protein upon Ligand
Binding--
Reconstitution of the heavy
chain-
2-microglobulin-ligand heterotrimer was achieved
by dialysis (cellulose ester tubings, 500-Da cut-off) of a solution
containing 0.15 mg/ml heavy chain, 0.1 mg/ml
2-microglobulin, and 0.1 mg/ml peptide ligand, using 5 mM glutathione to establish reducing conditions in 6 M urea against TBS. The solution was sparged with nitrogen
to prevent premature formation of disulfide bridges and oxidation of
free Cys67 in the B*2705 heavy chain. After 36-48 h at
10 °C, the mixture was concentrated to 500 µl in a Centripep
ultrafiltration unit (Amicon-Grace Ltd.). The folded heterotrimer was
purified by gel filtration on a superdex 75 column (Amersham Pharmacia
Biotech) with UV detection at 280 nm. The chromatogram showed three
major peaks at 9-, 11.5-, and 14-ml elution volume corresponding to heavy chain aggregates, refolded complex, and excess
2-microglobulin, respectively. The overall yield of the
fully reconstituted heterotrimer varied around 5%. The heterotrimer
peak was collected, concentrated in a Centricon 30 ultrafiltration unit
(Amicon-Grace), and immediately subjected to thermal denaturation.
Monitoring the Thermal Stability of MHC-Ligand Complexes by CD
Spectroscopy--
All CD measurements were done on a Jasco J-720
polarimeter with a water-jacketed 1-mm sample cell connected to a
computer-interfaced Neslab 111 circulating water bath. Temperature
control was achieved by measuring the circulating water immediately
after the sample cell. The thermal denaturation profiles were recorded
at 218 nm in 10 mM Tris, 150 mM NaCl (pH 8.0)
with the Jasco TEMPSCAN software using 0.1 °C increments at a
heating rate of 30 °C/h. Sample concentrations were determined
photometrically and held at 0.2 mg/ml. Different scan rates did not
affect the Tm value of B*2705 in complex with a reference
peptide (GRAFVTIGK; compare Ref. 34 and Table II). Three denaturation
curves from independent refolding preparations were averaged, after
conversion to molar ellipticity values. The curves were reduced to 70 data points by replacing each of the 10 neighboring points with their
mean value. By assuming a two-state equilibrium (35), data were fitted
by a nonlinear least-squares routine with the program Origin 2.9 (MicroCal Software, Inc.) to the following equations.
|
(Eq. 1)
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(Eq. 2)
|
The measured ellipticity (
) is given as a function of the
temperature (T) with the enthalpy (
Hm), heat
capacity upon unfolding (
Cp), and the midpoint
temperature of unfolding (Tm) being the fitting parameters.
Initial estimates for
Hm were obtained by
plotting ln K versus 1/T (van't Hoff plot) in
the transition region.
Cp was assumed to be
temperature-independent (36), and initial values were estimated from
the primary sequence (37). The linear base-line functions of the
unfolded and folded states
u and
f were
determined as linear regressions of the pre- and post-transitional regions. The enthalpy change at the midpoint of unfolding
(
Hm) was determined by the least-squares fit of
the unfolding curve to Equation 1. Because
Cp
estimates obtained by this approach are not very accurate and the
Hm values are largely influenced by the observed
deviations from a two-state model, a direct extrapolation from the
midpoint of unfolding to obtain 
Gunfolding
at 25 °C was not taken into consideration.
 |
RESULTS |
Replacing a Pentapeptide with a Polymethylene Spacer in Four
Unrelated Natural Epitopes--
For mimicking the sequence of the
central pentapeptide part (P4-P8) of MHC-bound nonapeptides, any
nonpeptidic fragment needs first to reproduce as closely as possible
the conformation of this bulging part and second to allow the same
intermolecular distance between the neighboring anchoring positions (P3
and P9) that are linked by the new spacer. The key distance between
C-
atoms of P3 and P9 positions is 16.6 Å in the x-ray structure of
HLA-B*2705 complexed by a nonapeptide model (3). The same distance can
be easily obtained after linking a polymethylene chain (Aua, Fig.
1) to P3 and P9 residues by simple amide
bonds. The 11-amino undecanoate fragment was then chosen for its
optimal length in an extended conformation and the absence of any
substituents, which should allow a conformational flexibility
sufficient for a proper fit into the binding groove. To check the
independence of the proposed modification on the parent epitope
sequence, the Aua spacer was introduced in four unrelated sequences of
natural epitopes, known to bind to B*2705 (Table II). The question of whether the new ligands were able to remain tightly bound in the peptide binding cleft like the natural nonapeptides was addressed by
molecular dynamics simulations of the solvated complexes (Table II).
The computational protocol used has been previously shown to explain
the binding potency of several HLA-B27-binding peptides (38) and to
predict the high affinity of designed peptide analogues (20, 29).
Energy-minimized conformations show that the proposed bridging has
modified neither the overall conformation of the bound ligands nor the
intermolecular distance between P3 and P9 C-
atoms (Fig.
2). Moreover, the chemical substitutions
were compatible with the conservation of the main interactions between the altered peptides and B*2705, especially the electrostatic interactions provided by the two charged termini and the arginine found
at position 2 of B*2705-binding peptides (39, 40).

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Fig. 2.
Close-up into the binding groove of
HLA-B*2705 (orange surface) in complex with
peptides 7-10 (Table II). These structures represent
energy-minimized conformations obtained from the x-ray structure of
HLA-B*2705 in complex with a model peptide (Protein Data Bank entry
1hsa) under a previously described protocol (20, 29). The heavy chain
backbone atoms of HLA-B*2705 have first been fitted together in the
four complexes, and the protein atoms are not shown for the sake of
clarity. Since protein distortion upon energy minimization of the
resulting complexes is minimal, the MHC protein is here represented by
a unique molecular surface independent of the bound ligand. The color
coding is as follows: blue, nitrogen; red,
oxygen; white, carbon atoms of ligand 7; cyan,
carbon atoms of ligand 8; green, carbon atoms of ligand 9;
yellow, carbon atoms of ligand 10. The arrows
indicate two methyl substituents of the HB tetramer interacting with
the central pockets C/E of the protein. The figure has been
prepared using the program GRASP (51).
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In order to experimentally validate the proposed model, the four
B*2705-restricted T cell epitopes and their modified analogues (Table
II) were synthesized and then tested for their binding to B*2705, in an
in vitro epitope stabilization assay (30). Replacing the
central pentapeptide sequence by the unsubstituted Aua fragment led in
all cases to a slight decrease in B*2705 stabilization (Table II),
which was also reflected by a lesser thermal stability of the resulting
complexes monitored by CD spectroscopy (Fig. 3, A-D). The temperature
shift in the midpoint of unfolding depends on the sequence of the
reference peptide but varies from
7 to
14 °C (Table II). Whereas
the effect of the Aua spacer is similar in both assays, there seems to
be no clear correlation between the EC50 scores obtained
from the epitope stabilization assay and the Tm values
calculated from the thermal denaturation experiments. The
Tm values reported here are fairly similar to those found for B*2705-binding peptides by other groups (21, 34, 41). The highest
stability against temperature was found for complexes with peptides 5 and 7, which all present a lysine at P9. Less favored amino acids at P9
are Val (peptides 1 and 2) and especially Arg (peptides 3 and 4), which
gives by far the least stable complexes with B*2705 (peptides 1 and 3, respectively).

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Fig. 3.
Thermal denaturation, monitored by CD
spectroscopy at 218 nm, of HLA-B*2705 loaded with ligands 1 and 2 (A), ligands 3 and 4 (B), ligands 5 and 6 (C), and ligands 7 and 8 (D). The
arrows indicate the midpoint of unfolding (Tm) of
the B*2705 heavy chain.
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Binding of peptides 1-6 to a closely related HLA-B27 subtype (B*2704)
was also examined by the same in vitro stabilization assay.
B*2704 differs from B*2705 by two amino acid changes in the peptide
binding groove (Asp77 to Ser; Val152 to Glu),
which influence its peptide specificity, relative to B*2705 (42). In
contrast to B*2705, substitution of Aua spacers for P4-P8 dramatically
decreases binding to B*2704 in our epitope stabilization assay (Table
II) when the last anchoring position (P9) is a basic amino acid (Lys,
Arg). If P9 is an apolar residue (Val, peptide 2), no real change in
B*2704 binding was noticed.
Substituting 3-Hydroxybutyrate Oligomers for the P4-P8 Sequence of
a Natural Peptide--
The decreased binding of the Aua analogues to
B*2705 is probably due to the nonfunctionalized nature of the
introduced spacer and the lack of interactions between the
unsubstituted Aua moiety and the central part of the binding groove.
Thus, a rational improvement in terms of binding affinity would be to
ramify the spacing moiety in order to reach one of the two central
pockets (pockets C and E) of the peptide binding groove that face the
spacer fragment. The (R)-3-hydroxybutyrate (HB) monomer was
selected for three main reasons: (i) polymers of HB are chemically
stable (43); (ii) they adopt conformations whose folding in the free
state resembles that found for peptides (44); and (iii) the methyl substituent is large enough to fit into pockets C and E. Thus, a trimer
(three units) and a tetramer (four units) of HB were substituted for
the P4-P8 sequence of one natural peptide (polyesterpeptides 9 and 10;
Table II), since they should optimally span the key distance between
the two anchor positions (P3 and P9) to bridge (Fig. 1).
In order to circumvent cyclization of the N-terminal glutamine (45)
that would prevent binding of the peptidic N terminus in the A pocket
of B*2705, the Ala1 analogue was also synthesized in the
nonnatural series (polyesterpeptides 11 and 12; Table II).
The modified ligands 9-12 have totally different binding affinities in
the in vitro stabilization assay (Table II), the
tetramer-containing compounds (ligands 10 and 12) being about 15 times
more potent that the trimeric analogues (ligands 9 and 11).
Furthermore, a HB tetramer segment leads to a significant enhancement
of the binding to B*2705 relative to the natural pentapeptide sequence. Again, the differences observed between natural and polyesterpeptides in the in vitro stabilization assay are not reflected by the
thermal denaturation experiments, performed only for ligands 11 and 12. Both compounds promote a similarly high stability of the resulting MHC-ligand pair with Tm values of 62-63 °C (Fig.
4, Table II) analogous to that found for
the parent peptide 7, and characteristic of high affinity ligands
(41).

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Fig. 4.
Thermal denaturation, monitored by CD
spectroscopy at 218 nm, of HLA-B*2705 loaded with ligands 7, 11, and
12.
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Molecular Modeling of the Altered Peptides in Complex with
B*2705--
A rationale for the (de)stabilizing effects of the three
spacers presently studied is proposed by the molecular dynamics
time-averaged conformations of a reference peptide (QRLKEAAEK; peptide
7) and its analogues (peptides 8-10). By looking at all close
nonbonded contacts between any peptide residue and its protein
neighboring atoms, the three spacers (Aua, HB trimer, and HB tetramer)
can be easily distinguished (Fig. 5). The
Aua spacer provides fewer contacts to the MHC binding groove than the
pentameric P4-P8 sequence of the parent peptide 7. This could explain
the decreased binding affinity of Aua-containing peptides to B*2705.
The detrimental effect of the HB trimer can be explained by the
weakening of the interactions between both terminal residues (PN and
PC) and their respective pockets (A and F). The better complementarity
of the HB tetramer to the B*2705 binding cleft is probably related to the following factors: (i) the additional interactions provided by two
methyl groups of the tetrameric spacer itself and (ii) a higher number
of nonbonded contacts of all other MHC anchors (PN, P2, P3, and
PC).

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Fig. 5.
Nonbonded interactions between the HLA-B*2705
protein and ligands 7-10, measured on energy-minimized time-averaged
conformations obtained after 200-ps Molecular Dynamics simulations of
the corresponding solvated complexes. Protein-ligand contacts are
recorded for interaction distances up to 4 Å.
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The total buried surface area of the modified ligands 8-10 has been
maintained when compared with that of the parent peptide 7 (about 650 Å2; data not shown). However, the total accessibility of
the ligands in their bound state is different. It is reduced by 20%
for HB analogues with respect to the natural epitope 7 (from 500 to 400 Å2). The Aua compound 8, although slightly less potent,
has a much lower accessible surface area (250 Å2) due to
the lack of substituents in the spacing area.
 |
DISCUSSION |
Replacing the central TcR-binding residues of MHC class I-bound
peptides (P4-P8) by nonpeptidic moieties has been reported previously
(20, 21). Herewith, we propose to rationalize the effect of three novel
spacers on binding to the HLA-B*2705 protein. The simplest spacer (Aua)
is a single polymethylene chain linking the P3 and P9 positions by
amide bonds. In accordance with a previous report studying the effect
of non-
amino acids (20), the Aua spacer does not impair binding to
B*2705. Only a moderate decrease in relative binding to B*2705 was
observed in an epitope stabilization assay, performed for four
unrelated modified peptides (Table II). However, the effect of this
modification on the thermal stability of the resulting MHC-ligand pair
was more significant (Fig. 3, A-D). Depending on the
peptide in which the Aua moiety was introduced, the midpoint of
unfolding of the B*2705 heavy chain (Tm) was lowered
by 7-14 °C. The corresponding free energy change in unfolding

Gunfolding at the midpoint of unfolding,
derived from the CD spectra (22), varies from
0.8 to
1.3 kcal/mol.
Since unfolding of the heavy chain should follow release of the ligand,
this observation supports a faster dissociation of the modified
peptides with respect to the parent epitope, as recently illustrated in
a homogeneous series of H-2Kd-binding nonapeptides (46).
However, the present study suggests that extrapolating peptide binding
differences from Tm values is not allowed for unrelated
sequences. For the set of 4 T cell epitopes presently studied,
EC50 values cannot be related to melting temperatures
calculated by CD spectroscopy. A likely explanation for this is that
binding, as measured in epitope stabilization assays, is significantly
influenced by the association rate of the peptide, whereas CD
measurements relate only to the dissociation rates. The highest thermal
stabilities were obtained for the B*2705 protein in complex with
peptide ligands bearing a Lys at P9. This makes sense, since Lys is the
P9 residue most complementary to its binding pocket F. Its side chain
forms a buried salt bridge with Asp116, located at the
bottom of the pocket. The predominance of the enthalpic contribution to
peptide dissociation would thus be compatible with the lower
Tm values observed with peptides having an amino
acid (Val, Arg) for which the interaction with pocket F is weaker. It
also corroborates previous computational simulations, suggesting that
peptide dissociation first occurs at the C terminus (20, 29, 38).
Interestingly, the effect of the Aua spacer is
subtype-dependent, since differences between the natural
and the Aua peptides in binding to B*2704 were much more significant
(Table II). B*2704 basically differs from the B*2705 allele by its weak
propensity to present peptides with basic P9 amino acids and its
improved suitability for nonpolar P9 residues (42). Thus, the
deleterious effect of the Aua spacer is amplified for peptides bearing
a weak anchoring amino acid at P9 (peptides 4 and 6; Table II) and
decreased for peptides with nonpolar P9 residues (peptide 2). The Aua
group can be considered as a monofunctional spacer, since it simply provides the covalent linkage between two neighboring anchor positions (P3 and P9). Therefore, it has the same effect on HLA binding as
previously reported spacing moieties like oligomers of 4-aminobutyrate or 6-aminohexanoate (20) or substituted phenanthridines for which a
similar thermal destabilization (
Tm of
12 °C) has been reported (21). However, a modification of
TcR-binding amino acids that also enhances the binding affinity for the
host MHC protein is possible. We describe here the first bifunctional spacer that provides additional interactions to the binding groove. The
tetramer of HB, introduced between P3 and P9, significantly enhances
binding to B*2705 (Table II). The beneficial effect of the
(HB)4 spacer is attributed to two of its methyl
substituents that reach the central pockets C/E of the binding cleft
(Fig. 2). Since the global binding mode of the modified peptide has not
been altered, the direct consequence of this replacement is an enhanced
number of nonbonded contacts with the protein (Fig. 5). Again,
discrepancies are observed for that series of compounds (ligands 7-12)
between EC50 values and melting temperatures derived from
CD experiments on the reconstituted complexes (Fig. 4). Tm values calculated for the tetrameric and trimeric HB analogues are
nearly identical, whereas a 12-16-fold decreased binding was observed
after shortening the length of the spacing area by one HB unit. The
Tm values of a series of MHC-peptide complexes have recently
been directly related to experimental equilibrium dissociation
constants, KD (46). Thus, the higher affinity observed for the (HB)4 compounds relative to the parental
peptide and the trimeric analogues could be due to faster on-rate
kinetics. Alternatively, since the correlation proposed by Morgan
et al. (46) takes into account a series of highly related
nonapeptides, it may not be valid for altered ligands lacking a
canonical nonapeptide structure. Importantly, the present study
demonstrates that CD denaturing curves cannot be used alone to explain
differences in binding of altered peptide ligands to a class I MHC
protein. This is of crucial importance in any design effort aimed at
enhancing binding affinities by increasing the on-rate kinetics of the
designed molecule. It should be noted that two CD denaturation curves
(peptides 7 and 12, Fig. 4) slightly deviate from the expected
two-state model by presenting an additional transition at a temperature (45 °C) corresponding to the unfolding of peptide-free heavy chain (47). Such deviations from an ideal two-state model have already been
observed (34) but remain difficult to explain at the molecular level.
Our data demonstrate that B*2705-restricted epitopes may be easily
modified by introducing simple nonpeptidic elements in their central
part without drastic changes in binding to their restriction MHC
proteins. Two conditions seem to be necessary for these modifications:
(i) the last amino acid (PC) should be a strong anchor, and (ii) the
parent epitope should not contain a dominant anchor position between
the P4 and P8 positions. Since this is the case for a majority of class
I MHC peptide binding motifs (8), such chemical manipulations should be
feasible for many antigenic peptides binding to class I MHC proteins.
Class II MHC-binding peptides that utilize nearly all peptidic bonds to
interact with their host MHC protein (48) must be excluded from these
epitope modifications.
The altered ligands reported in this study constitute a further step
toward obtaining full nonpeptide ligands for class I MHC proteins. They
represent interesting tools for altering the response of
B*2705-restricted T cells to naturally occurring antigenic peptides and
for designing novel synthetic vaccines.
We thank the calculation center of the ETH
Zürich for allocation of computer time on the CRAY J90 and
PARAGON supercomputers.