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*
Department of Molecular and Cellular Biology and
Howard Hughes Medical Institute, Harvard University, Cambridge MA 02138;
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093; and
§
Molecular Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Tm = 16°C, than the Tax9 complex,
but both can sensitize target cells for lysis by some Tax-specific CTL
from HTLV-1 infected individuals. The absence of a P1 peptide residue
is thus not enough to prevent formation of a "closed conformation"
of the peptide-binding site. TCR affinity measurements and cytotoxic T
cell assays indicate that the Tax8/HLA-A2 complex does not functionally
cross-react with the A6-TCR-bearing T cell clone specific for
Tax9/HLA-A2 complexes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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9 residues) from proteins degraded in the cytoplasm of
vertebrate cells are bound by class I MHC molecules in the endoplasmic
reticulum and transported to the cell surface for recognition by the
Ag-specific receptors (TCR) of T cells as part of the immune system’s
surveillance for foreign Ags (1, 2). Any one class I
molecule, of the many different alleles expressed in the population, is
capable of forming very stable complexes, with half-lives of tens of
hours, with a large number of different short peptides. These
long-lived MHC/peptide complexes, which can mark cells for destruction
by CTL, appear to be kinetic traps for peptides. In vitro, the
off-rates of peptides from MHC molecules are very slow (3, 4), whereas peptide association rates vary between experimental
systems (5, 6, 7, 8, 9). The kinetics of MHC assembly suggests a
two step process involving a conformational change of the MHC molecule
from a short-lived, receptive, "open" binding state to a
long-lived, "closed" conformation (5, 10, 11, 12, 13). X-ray crystal structures of MHC molecules have revealed the structure of the closed conformation with peptides bound (14, 15, 16). Some side chains of bound peptides (anchor residues) are held in pockets in the peptide-binding groove that are polymorphic in the different MHC allelic products, providing a sequence-dependent element to peptide binding (16, 17, 18). In class I MHC molecules the charged N and C termini and main chain of the bound peptide are held, through a network of hydrogen bonds and salt bridges, to polar residues conserved in all human and murine class I MHC molecules (19, 20, 21, 22, 23, 24). This network of conserved hydrogen bonds at both termini of peptides provides an independent peptide sequence-independent element to peptide binding. Because these peptide N- and C-terminal contacts to class I MHC molecules are conserved for both all bound peptides and all class I alleles, whereas the peptide side chain contacts to the polymorphic pockets of class I molecules vary considerably with different peptides and different MHC alleles, the conserved interactions at the peptide termini have been proposed to have an important role in forming the shared property of long half-life peptide-bound conformations of class I molecules (19).
The stability of class I MHC molecules, which are heterodimers of a
polymorphic heavy chain
(Hc)3 and
ß2-microglobulin (ß2m),
is strongly dependent on the presence of a bound peptide. In vitro, in
the absence of bound peptides, ß2m dissociates
and Hc aggregates (17, 22, 25, 26, 27, 28, 29), whereas in vivo in the
endoplasmic reticulum peptide-free class I molecules are stabilized by
chaperonins and the peptide transport and loading proteins, TAP and
Tapasin (30, 31). Thermal denaturation studies of
MHC/peptide complexes in which either the peptide N-terminal amino
group or C-terminal carboxylate group was substituted by a methyl group
showed a decrease in the Tm of the
MHC/peptide of 22°C, indicating a decrease in stability of 4.6
kcal/mol, whereas substituting both peptide anchor residues with
alanine showed a decrease of only 5.5°C or 1.2 kcal/mol (28, 32). These thermodynamic data support the suggestion that the
conserved interactions between the N and C termini of bound peptides
may dominate in the formation and stabilization of peptide/MHC
complexes.
In this paper we have studied the effect of removing the N-terminal
amino acid of an antigenic peptide (Tax9) on the stability and
structure of its interaction with HLA-A2, and on its recognition by T
cells as an HLA-A2/Tax8 complex. Tax9 is the dominant antigenic peptide
inducing cytotoxic T cells in human T cell lymphotrophic virus-1
(HTLV-1)-infected individuals with the neural degenerative disorder
HAM/TSP (33). The x-ray structure of the HLA-A2 complex
with Tax9 (34) was previously refined to 2.5-Å
resolution, whereas the complexes of HLA-A2/Tax9 with two human
ßTCRs, A6 and B7, were determined at 2.6-Å resolution
(35, 36, 37). Although HLA-A2/Tax8 activates cytotoxic T cells
in HTLV-1-infected individuals (38), we find that it does
not cross-react functionally with HLA-A2/Tax9-specific T cells in cell
lysis assays and has a low affinity for a HLA-A2/Tax9-specific TCR. The
stability of the HLA-A2/Tax8 complex is markedly reduced, as expected
for loss of the conserved interactions at the N-terminal
peptide-binding site, but the structure of the complex is remarkably
similar with water molecules substituting for some of the peptide
interactions in the binding site.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The extracellular region of the HLA-A2 Hc and
ß2m were expressed separately in
Escherichia coli as inclusion bodies (39). The
inclusion bodies were refolded together in the presence of excess Tax8
(LFGYPVYV) or Tax9 (LLFGYPVYV) peptide. Briefly, milligram amounts of
ß2m, Hc, and peptide were injected into 500 ml
of a refolding buffer (100 mM Tris, 400 mM arginine-HCl, 2 mM NaEDTA,
0.5 mM oxidized glutathione, and 5 mM reduced glutathione, pH 8.3). The
final concentrations of the constituents of the complexes were 2 µM
ß2m, 1 µM Hc, and 50 µM peptide (Tax9). A
significantly larger excess of Tax8 peptide (100–150 µM) was
required to produce that complex in sufficient quantity for biochemical
studies, and the resulting yield was very low (5%) compared with
typical yields with Tax9 (20–25%). The ternary complexes were
purified by ion-exchange and gel filtration chromatography as described
previously (34).
Crystals of Tax9-A2 were obtained by vapor diffusion from hanging drops containing equal volumes of protein (5 mg/ml; 25 mM MES, pH 6.5) and 13–20% polyethylene glycol (PEG) 6000 (25 mM MES, pH 6.5). Tax8-A2 crystals were obtained by seeding with Tax9-A2 crystals. A 3-µl solution of seed crystals was incubated with a 3-µl solution containing 3 mg/ml Tax8-A2 and 400 µM Tax8 peptide in 25 mM MES (pH 6.4) and 0.1% NaN3. The drop was equilibrated against 13% PEG 6000, 25 mM MES, and 0.1% NaN3 at 18°C.
Data collection and structural refinement
Crystals of Tax8-A2 were transferred to a 20% glycerol solution in steps of 4–10% and flash-cooled in a stream of cryo-cooled nitrogen gas. Data were collected on a Mar345 detector (Mar Research, Hamburg, Germany) mounted on an Elliot GX-13 x-ray generator (GEC Avionics, London, U.K.). The structure of Tax8-A2 was refined by using the previously determined structure of Tax9-A2 as the starting model (Protein Data Bank code 1hhk; Ref. 34). A subset (10%) of the reflection data were flagged (R-free) and excluded from refinement protocols. As the crystals of Tax8-A2 and Tax9-A2 were isomorphous, the same set of flagged reflections previously used for monitoring Tax9-A2 refinement was used in the R-free data set for Tax8-A2. Because the structure of Tax9-A2 was previously refined to only 2.5-Å resolution (without waters), the data were extended to 1.8 Å and the model refined to that resolution with the inclusion of water molecules.
The starting model of Tax8-A2, stripped of water molecules and the
peptide was initially subjected to rigid-body fitting using the program
CNS (40). Electron density maps clearly indicated the
position of Tax8 within the peptide-binding groove of HLA-A2. The
structure was further refined by multiple cycles of energy minimization
and model building using the programs CNS and O (40, 41).
All reflections between 50 Å and the resolution limits of the data
sets (2.14 Å for Tax8-A2, 1.8 Å for Tax9-A2) were used during
refinement and electron density map calculations (Table I
). Water molecules were gradually
introduced during the course of model building and were selected from
>3
peaks in difference (Fo -
Fc) electron density maps within
2.5–3.6 Å of hydrogen bond donors or acceptors.
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-amino group of Tax8. Simulated annealing
omit maps, in which the peptide and surrounding water molecules were
removed, confirmed that the N terminus was both well positioned and
ordered in both molecules of the asymmetric unit. The conformation
of Leu2 precludes formation of a salt bridge with
Glu63 of the B pocket; in the Tax9 structure, the
identical amide nitrogen hydrogen bonds to Glu63.
To further test the validity of the Tax8 model, one further cycle of
positional refinement was performed in which the -amino group was
manually re-fitted by adjustment of torsion angles to within 3.2 Å of
the side-chain of Glu63 (O
1). Following
crystallographically restrained energy minimization in the CNS program,
it was again observed that the N terminus shifted away, to a position
3.6 Å from Glu63 (O1), but to within 3.29 Å
of the hydroxyl group of Tyr159. The statistics
from the final cycle of refinement are shown in Table II. Coordinates and structure factors
have been submitted to the Protein Data Bank (A2-Tax8 and A2-Tax9 PDB
codes 1DUY and 1DUZ, respectively).
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The thermal stability of peptide-MHC complexes was monitored by
CD spectroscopy using a Jasco J-710 instrument (Jasco International,
Tokyo, Japan) equipped with a Peltier temperature regulator. Solution
conditions were 20 mM phosphate and 75 mM NaCl (pH 7.4). Protein
concentrations were 0.15 mg/ml. The spectrum between 250 and 190 nm
indicated a predominantly ß-sheet conformation. Temperature
denaturation was monitored at the minimum of 218 nm between 10 and
90°C using a gradient of 1°C/min. Scans were repeated twice with
fresh protein and the data points averaged. Rather than constraining
the data to a two-state unfolding model, the data in the transition
region were simply fit to a nine-order polynomial equation, and the
apparent Tm was determined from the
maximum of the first derivative of the fitted curve.
Binding measurements
The 2G4 T cell clone has a TCR (A6-TCR) specific for Tax9
complexed with HLA-A2, and the interaction of this receptor with a
number of altered peptides complexed with HLA-A2 has been previously
investigated (37, 42). The binding of Tax8-A2 to the
A6-TCR was investigated here using an equilibrium BIAcore assay as
described previously (37). Briefly, recombinant A6-TCR
with a single free thiol at the C terminus of the ß-chain was coupled
to a CM5 sensor chip using standard thiol coupling. Multiple
concentrations of Tax8-A2 were injected at a flow rate of 10 µl/min,
and the responses at equilibrium (400 s after injection) were
determined. The temperature of the sample was maintained at 4°C, and
Tax8-A2 dilutions were made from a highly concentrated stock
immediately before injection to minimize dissociation of the Tax8
peptide from HLA-A2. The responses from injections over a mock surface
were subtracted from the data, and all injections were repeated twice.
Equilibrium responses were fit against the concentration of injected
Tax8-A2, assuming a single-site binding model. Solution conditions were
10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% polysorbate-20 (pH
7.4) at 25°C.
T cell assays
T cell-mediated cytotoxicity was quantitated by a time-resolved fluorometric assay using HLA-A2-transfected cells as targets as previously described (36). Effector cells were the A6-TCR-expressing CD8+ T cell clone 2G4 that recognizes the Tax11–19 peptide presented by HLA-A2 and was isolated from a patient with HAM/TSP (43).
For the antagonism assay, HLA-A2-transfected Hmy2.C1R cells were
treated with 100 µg/ml mitomycin C (Sigma, St. Louis, MO) for 2
h at 37°C, washed three times with PBS, and then pulsed with 1000 nM
of candidate antagonist peptides for 2 h at 37°C. The cells were
washed, and 1 x 105 cells were incubated
with 1 x 105 2G4 T cells for 1 h at
37°C. Tax peptide was added at concentrations of 0.1–10 nM, and the
cells were incubated for 48 h. Supernatants were collected and
assayed for IFN-
content as previously described
(36).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The structures of Tax8-A2 and Tax9-A2 were refined to reasonable
agreement between observed and calculated structure factors, as well as
good stereochemistry and geometry (Table II
). Although data extending
to 2.15 Å were used in the refinement of Tax8-A2, the data were
incomplete in the highest resolution shell (Table I). The number of
observed reflections corresponds to a 100% complete data set to
2.30-Å resolution. The overall structure of the Tax8-A2 complex is
identical to Tax9-A2 and is shown in Fig. 1. The quality of the structures permits
a detailed discussion of the peptide-binding groove and its bound
solvent.
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). Hydrogen bonds and hydrophobic
contacts are preserved from Phe3 to
Val9 of Tax8 and the corresponding MHC binding
pocket, indicating that loss of the position 1 Leu (P1) does not induce
global shifts or conformational changes in the peptide or MHC.
Following alignment of the HLA Hc, the root-mean-square difference in
the coordinates of the common atoms of Tax8 and Tax9 was 0.36 Å.
However, the -amino group of Leu2 (Tax8) is
positioned about 1 Å from the equivalent P2 amide nitrogen of Tax9
(see Materials and Methods), making a hydrogen bond with the
hydroxyl of Tyr159 rather than
Glu63 as in Tax9 (compare Fig. 3, a and b). The
distance between the side chain of Glu63 and the
P2 amino group of Tax8 is 3.9 Å, which may still permit a favorable
electrostatic interaction.
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In the Tax8 complex two water molecules, Wat-1 and Wat-2,
partially fill the space occupied by the P1 peptide residue,
Leu1, in the Tax9 complex (Fig. 3). Wat-1
provides a bridge from the N terminus of Tax8, via hydrogen bonds, to
the carboxylate group of Glu63 (Fig. 3). Wat-2 is
in the position that is occupied by the -amino group of
Leu1 in Tax9 (Fig. 3, a and
b). This water provides a bridge from the hydroxyl group of
Tyr7, in the ß-sheet forming the floor of the
peptide-binding groove, via hydrogen bonds to
Tyr171 in -helix forming one side of the
binding site (Fig. 3). In the Tax9 complex, the same hydrogen bonded
bridge between elements of secondary structure is made through the
amino group at Leu1 of the bound peptide (Fig. 3b). Wat-2 is also within hydrogen bonding distance of Wat-1
(distance of 2.82 Å; Fig. 3a) and a third water (Wat-3)
found in both the Tax8 and Tax9 complexes (Fig. 3).
The hydrogen bonds in the peptide N-terminal region of the binding
groove for the Tax8-A2 and Tax9-A2 structures are summarized in Table III. Although in the Tax8 complex there
is a loss of three hydrogen bonds that went directly from the bound
peptide to the MHC molecule relative to Tax9 complex, the shift in the
nitrogen position of Tax8 and the addition of two water molecules
creates a network of hydrogen bonds that both satisfies all of the
hydrogen bonding acceptors and donors of the conserved MHC residues and
cross-links together the same secondary structure elements as in the
Tax9 complex.
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Solvent molecules in the peptide-binding sites of both Tax8 and Tax9 complexes
In the middle portion of the peptide, three buried water molecules
form part of the interface between Tax8 and Hc (Wat-4, Wat-5, Wat-6;
Fig. 4). These waters are also present in
the structure of Tax9-A2 refined here, but were not visible in the
previously published, lower resolution (2.5 Å) structure
(34). The carbonyl oxygen of Tyr5 is
linked via Wat-4 to the -amino group of Arg97.
The carbonyl oxygen of Val7 is linked via Wat-6
to the carboxylate oxygen of Asp77. These
water-mediated, hydrogen bond bridges are formed with the part of the
peptide that is central to recognition by TCRs (35, 37).
Upon binding of both the A6- and B7-TCRs, a conformational change takes
place in the Tax9 peptide such that the side chain of
Pro6 becomes buried in the pocket occupied by two
of these water molecules (35), and the
Val7 side chain projects outward toward the TCR.
Consequently, two water molecules, Wat-4 and Wat-5, must be displaced
upon TCR binding. Another water molecule, Wat-7, bridges the C-terminal
carboxylate of the peptide via hydrogen bonds to the side chain of
Thr80 (O1) in both the Tax8-A2 and Tax9-A2
structures.
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The thermal denaturation curves of Tax8-A2 and Tax9-A2 were
determined by monitoring the loss of secondary structure using CD. The
mid-point of the transitions were 49°C for Tax8-A2 and 65°C for
Tax9-A2 (Fig. 5), indicating a
significant destabilization of the peptide-A2 complex following loss of
the N-terminal residue of the peptide. A second transition observed
in the denaturation curves beginning near 80°C corresponds to the
unfolding of ß2m (27, 32). Consequently, the early loss
of secondary structure observed for Tax8-A2 is attributed to loss of
the peptide and unfolding of the Hc, as previously shown (27, 32). Due to linkage between peptide binding and stable folding
of the Hc/ß2m heterodimer, it follows that Tax8 binds the A2
molecule with a much lower affinity than Tax9 (28, 44). These results are consistent with peptide-binding studies
of the murine class I MHC H2-Kd, in which
deletion of the N-terminal residue of a nonomer peptide significantly
reduced the stability of the MHC/peptide complex (45).
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Binding affinity of Tax8-A2 to the A6-TCR, specific for
HLA-A2/Tax9, was measured using an equilibrium plasmon resonance
experiment. The A6-TCR clone, isolated from a patient with the
HTLV-1-associated autoimmune disorder HAM/TSP, is a class I-restricted
TCR specific for Tax9 bound to HLA-A2 (43). Equilibrium
binding experiments (Fig. 6A)
revealed that the association of the A6-TCR with Tax8-A2 is about
16-fold weaker (KD = 15 ± 2
µM) relative to the full-length Tax9 peptide-MHC complex
(KD = 0.9 ± 0.1 µM; Ref.
37). Although we attempted to minimize such contributions
during the experiment, it is possible that this result is influenced
slightly by dissociation of Tax8 from the MHC (see Materials and
Methods).
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B). The data show that,
compared with the wild-type Tax9 peptide, Tax8 has a very limited
ability to activate A6-TCR-bearing T cells (barely detectable lysis at
a million-fold increase in peptide concentration). In addition, no TCR
antagonism (vs Tax9) was detected with Tax8 peptide (Fig. 6C). However, Tax8 could sensitize targets cells for lysis
by some Tax-specific CTL (38). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Until now, all the structures of class I MHC molecules with
bound peptides determined by x-ray crystallography, whether the
peptides were 8-mer, 9-mer, or 10-mer, have had a peptide residue in
the P1 binding site making the conserved array of hydrogen bonds to
nonpolymorphic MHC residues (Fig. 3b). In the human class I
molecule HLA-B35, a short peptide was accommodated by stretching out a
kink usually found in longer peptides (46), and in murine
class I molecules the shape of the bottom of the binding site is
conducive to the binding of shorter peptides (21, 47). The
Tax8/Tax9 pair of peptides were studied here, because it seemed likely
that the 8-mer would bind just like the 9-mer, except with the P1 site
empty. This offered the opportunity to see whether the loss of the
conserved hydrogen bonds, which both held bound peptides in the site
and tethered the secondary structures of the binding site together,
would affect the conformation of the binding site. In particular, we
hoped to see whether the binding site might adopt a partially "open
state" as proposed to exist from the slow binding and dissociation
kinetics of peptides.
In the Tax8 complex, as a result of no peptide residue occupying the P1
binding site, we observed a loss of three hydrogen bonds directly
from the bound peptide to the MHC molecule, relative to the Tax9
complex. However, the shift in the primary amine position of Tax8, and
the addition of two water molecules, created a new network of hydrogen
bonds that satisfies all of the hydrogen bonding acceptors and donors
of the conserved MHC residues (Fig. 3). Furthermore, the new network of
hydrogen bonds in the Tax8 complex cross-links together the same
secondary structure elements as in the Tax9 complex. Thus, although the
structure indicates that the Tax8 peptide might be expected to bind
more weakly than Tax9, due to the loss of the three conserved hydrogen
bonds from peptide to MHC, the restoration of a hydrogen bonding
network using water molecules apparently serves to stabilize the
"closed conformation" of the MHC binding site.
In previous studies with a different peptide, the influenza virus
matrix peptide (GILGFVFTL), the N-terminal amino group of the peptide
was replaced, synthetically, by a methyl group (32). The
modified N terminus prevented the formation of two of the hydrogen
bonds that group normally makes with conserved residues of the MHC
molecule (28). This loss of interactions resulted in a
decrease of the thermal denaturation temperature of the peptide/HLA-A2
complex of 21°C, corresponding to a G° value of about 4.6
kcal/mol (28, 32). A structure of the complex revealed
that the substituted methyl group rotated away from the hydrogen
bonding groups of the MHC molecule and was replaced by a water molecule
occupying the position vacated by the peptide N terminus
(28) and located identically to water Wat-2 observed in
the Tax8 complex (Fig. 3a). The bound water molecule formed
a similar set of hydrogen bonds to the conserved MHC residues as the
full-length 9-residue matrix and Tax peptides, but did not provide any
bridging hydrogen bonds to the modified peptide. Apparently the loss of
these two hydrogen bonds to the N-terminally modified peptide resulted
in the large (4.6 kcal/mol) decrease in stability of the peptide/MHC
complex.
The thermal denaturation temperature of the Tax8 complex was
observed in this paper to be 16°C lower than that of the Tax9
complex. This difference also presumably results partly from the loss
of direct hydrogen bonds between the peptide and MHC molecule. Although
the bridging water molecules Wat-1 and Wat-2 form a network of hydrogen
bonds, the new hydrogen bonds do not directly link the peptide to the
MHC molecule (Fig. 3a). Thus, as in the earlier study of the
modified matrix peptide/MHC complexes, eliminating the conserved
hydrogen bonds from the peptide N terminus to the MHC molecule in the
Tax8 complex also very substantially decreased the stability of the MHC
molecule. In both cases, despite drastic changes in the stability of
the MHC molecules, when hydrogen bonding interactions with the peptide
were removed, the conformation of the peptide-binding site of the
destabilized molecules was not changed. Instead, in both cases, water
molecules were observed to bind and created networks of hydrogen bonds
that apparently maintained the binding site structure. This suggests
the possibility that even an empty binding site, devoid of peptide,
which is quite unstable relative to peptide/MHC complexes
(27), might maintain this closed conformation as the
result of binding water molecules at its ends. "Closed-empty" and
"closed-full" binding sites may look similar and dominate at
equilibrium, with "open-empty" and "open-full" states being
more transient states separated from the closed states by the high free
energy barriers responsible for the slow association and dissociation
kinetics. It is these transient states that enzyme-like molecules must
stabilize in vivo to accelerate peptide loading.
TCR binding and activation
HTLV-1 Tax8 peptide-specific HLA-A2-restricted CTL cell lines have been generated from PBL of patients with HTLV-1-associated neurological disease (38). The x-ray structure determined here of the Tax8/HLA-A2 complex establishes the P1 peptide pocket is empty, so that the TCR of Tax8-specific CTL must bind to a peptide/MHC complex with no P1 peptide side chain.
The x-ray structures of two TCR specific for Tax9/HLA-A2 have been
determined complexed with Tax9/HLA-A2 (35, 36, 37). In both
cases the major contacts between TCR and peptide are with peptide
residues P4 to P8, but the TCR does cover and make one atomic contact
to the P1 sidechain (Leu1). If we model a TCR
interaction with Tax8/HLA-A2 by superimposing the MHC/peptide
coordinates from the Tax8/MHC structure with the Tax9/MHC/TCR
structure, then the solvent accessible surface area of the MHC/peptide
buried by the TCR would be decreased by 194 Å2
(20%) as the result of deleting P1. However, a Tax8-specific TCR might
fill in this cavity with either a different CDR1 sequence or
conformation.
In published studies, it is the kinetic off-rate of the TCR from the
MHC/ligand complex that most often correlates with the nature of the T
cell response (48). The kinetic off-rate of A6-TCR with
Tax9/HLA-A2 is 0.093 s-1
(37, 42), in the range of a typical agonist. The kinetic
off-rate of the A6-TCR with Tax8/HLA-A2 was too fast to measure by
surface plasmon resonance, consistent with the extremely low T cell
response observed (Fig. 6, B and C).
The affinity for a Tax9 specific ßTCR ectodomain for the
Tax8/HLA-A2 complex was measured as 16-fold lower than its affinity for
Tax9/HLA-A2 (Fig. 6A). This affinity difference is at least
qualitatively consistent with the modeling that indicates a potential
cavity in the interface. A 16-fold magnitude of affinity difference in
other TCR/peptide/MHC complexes has been observed to be sufficient to
alter T cell responses dramatically (37, 42, 49, 50).
Qualitatively consistent with this expectation, we observed markedly
reduced (>106 reduction) activity in
Tax8-induced cell lysis of a Tax9-specific T cell line (Fig. 6B) and no antagonism of Tax9 activity at 1 µM Tax8
peptide concentration (Fig. 6C). Quantitatively, however,
the absolute affinity, KD = 15 ±
2 µM, of A6-TCR for the Tax8/HLA-A2 ligand, is in the range that
often either signals as a partial agonist or an antagonist (37, 42),
rather than as a null as observed here. This comparison suggests that
the Tax8/HLA-A2 complex may have unusual properties as a cell surface
ligand for a T cell. Apparently the decreased stability of the Tax8/MHC
complex (Tm = 49°C), coupled with
the 16-fold decrease in TCR affinity, combine somehow to decrease the
effectiveness of the Tax8/MHC complex as a ligand in cellular assays
(since neither alone prevents either partial agonist or antagonist
responses).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Don C. Wiley, Howard Hughes Medical Institute, Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138.
3 Abbreviations used in this paper: Hc, heavy chain; ß2m, ß2-microglobulin; Tm, melting temperature; HTLV-1, human T cell lymphotrophic virus-1; HAM/TSP, HTLV-I-associated myelopathy/tropical spastic paraparesis; CD, circular dichroism.
Received for publication January 28, 2000. Accepted for publication March 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501. Immunity 4:203.[Medline]
ß T cell receptors. Annu. Rev. Immunol. 16:523.[Medline]
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 S. Wan, P. V Coveney, and D. R Flower Peptide recognition by the T cell receptor: comparison of binding free energies from thermodynamic integration, Poisson-Boltzmann and linear interaction energy approximations Phil Trans R Soc A, August 15, 2005; 363(1833): 2037 - 2053. [Abstract] [Full Text] [PDF]
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S. Wan, P. V. Coveney, and D. R. Flower Molecular Basis of Peptide Recognition by the TCR: Affinity Differences Calculated Using Large Scale Computing J. Immunol., August 1, 2005; 175(3): 1715 - 1723. [Abstract] [Full Text] [PDF]
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O. Y. Borbulevych, T. K. Baxter, Z. Yu, N. P. Restifo, and B. M. Baker Increased Immunogenicity of an Anchor-Modified Tumor-Associated Antigen Is Due to the Enhanced Stability of the Peptide/MHC Complex: Implications for Vaccine Design J. Immunol., April 15, 2005; 174(8): 4812 - 4820. [Abstract] [Full Text] [PDF]
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 C. S. Clements, L. Kjer-Nielsen, L. Kostenko, H. L. Hoare, M. A. Dunstone, E. Moses, K. Freed, A. G. Brooks, J. Rossjohn, and J. McCluskey Crystal structure of HLA-G: A nonclassical MHC class I molecule expressed at the fetal-maternal interface PNAS, March 1, 2005; 102(9): 3360 - 3365. [Abstract] [Full Text] [PDF]
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M. Probst-Kepper, H.-J. Hecht, H. Herrmann, V. Janke, F. Ocklenburg, J. Klempnauer, B. J. van den Eynde, and S. Weiss Conformational Restraints and Flexibility of 14-Meric Peptides in Complex with HLA-B*3501 J. Immunol., November 1, 2004; 173(9): 5610 - 5616. [Abstract] [Full Text] [PDF]
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T. K. Baxter, S. J. Gagnon, R. L. Davis-Harrison, J. C. Beck, A.-K. Binz, R. V. Turner, W. E. Biddison, and B. M. Baker Strategic Mutations in the Class I Major Histocompatibility Complex HLA-A2 Independently Affect Both Peptide Binding and T Cell Receptor Recognition J. Biol. Chem., July 9, 2004; 279(28): 29175 - 29184. [Abstract] [Full Text] [PDF]
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A. J. H. Borysik, S. E. Radford, and A. E. Ashcroft Co-populated Conformational Ensembles of {beta}2-Microglobulin Uncovered Quantitatively by Electrospray Ionization Mass Spectrometry J. Biol. Chem., June 25, 2004; 279(26): 27069 - 27077. [Abstract] [Full Text] [PDF]
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A. I. Webb, M. A. Dunstone, W. Chen, M.-I. Aguilar, Q. Chen, H. Jackson, L. Chang, L. Kjer-Nielsen, T. Beddoe, J. McCluskey, et al. Functional and Structural Characteristics of NY-ESO-1-related HLA A2-restricted Epitopes and the Design of a Novel Immunogenic Analogue J. Biol. Chem., May 28, 2004; 279(22): 23438 - 23446. [Abstract] [Full Text] [PDF]
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L. Li and M. Bouvier Structures of HLA-A*1101 Complexed with Immunodominant Nonamer and Decamer HIV-1 Epitopes Clearly Reveal the Presence of a Middle, Secondary Anchor Residue J. Immunol., May 15, 2004; 172(10): 6175 - 6184. [Abstract] [Full Text] [PDF]
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L. Gales, I. Cardoso, B. Fayard, A. Quintanilha, M. J. Saraiva, and A. M. Damas X-ray Absorption Spectroscopy Reveals a Substantial Increase of Sulfur Oxidation in Transthyretin (TTR) upon Fibrillization J. Biol. Chem., March 21, 2003; 278(13): 11654 - 11660. [Abstract] [Full Text] [PDF]
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G. Denkberg, E. Klechevsky, and Y. Reiter Modification of a Tumor-Derived Peptide at an HLA-A2 Anchor Residue Can Alter the Conformation of the MHC-Peptide Complex: Probing with TCR-Like Recombinant Antibodies J. Immunol., October 15, 2002; 169(8): 4399 - 4407. [Abstract] [Full Text] [PDF]
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C. H. Trinh, D. P. Smith, A. P. Kalverda, S. E. V. Phillips, and S. E. Radford Crystal structure of monomeric human beta -2-microglobulin reveals clues to its amyloidogenic properties PNAS, July 23, 2002; 99(15): 9771 - 9776. [Abstract] [Full Text] [PDF]
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J. Yague, A. Marina, J. Vazquez, and J. A. Lopez de Castro Major Histocompatibility Complex Class I Molecules Bind Natural Peptide Ligands Lacking the Amino-terminal Binding Residue in Vivo J. Biol. Chem., November 16, 2001; 276(47): 43699 - 43707. [Abstract] [Full Text] [PDF]
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T. M. C. Hornell, S. M. Martin, N. B. Myers, and J. M. Connolly Peptide Length Variants p2Ca and QL9 Present Distinct Conformations to Ld-Specific T Cells J. Immunol., October 15, 2001; 167(8): 4207 - 4214. [Abstract] [Full Text] [PDF]
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P. Sliz, O. Michielin, J.-C. Cerottini, I. Luescher, P. Romero, M. Karplus, and D. C. Wiley Crystal Structures of Two Closely Related but Antigenically Distinct HLA-A2/Melanocyte-Melanoma Tumor-Antigen Peptide Complexes J. Immunol., September 15, 2001; 167(6): 3276 - 3284. [Abstract] [Full Text] [PDF]
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B. E. Gewurz, R. Gaudet, D. Tortorella, E. W. Wang, H. L. Ploegh, and D. C. Wiley From the Cover: Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2 PNAS, June 5, 2001; 98(12): 6794 - 6799. [Abstract] [Full Text] [PDF]
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 B. M. Baker, R. V. Turner, S. J. Gagnon, D. C. Wiley, and W. E. Biddison Identification of a Crucial Energetic Footprint on the {{alpha}}1 Helix of Human Histocompatibility Leukocyte Antigen (HLA)-A2 That Provides Functional Interactions for Recognition by Tax Peptide/HLA-A2-specific T Cell Receptors J. Exp. Med., February 26, 2001; 193(5): 551 - 562. [Abstract] [Full Text] [PDF]
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A. K. Sharma, J. J. Kuhns, S. Yan, R. H. Friedline, B. Long, R. Tisch, and E. J. Collins Class I Major Histocompatibility Complex Anchor Substitutions Alter the Conformation of T Cell Receptor Contacts J. Biol. Chem., June 8, 2001; 276(24): 21443 - 21449. [Abstract] [Full Text] [PDF]
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) and Tax8 (
) to lysis by the A6-TCR bearing CTL
clone 2G4 (E:T = 2.5:1). A control peptide (influenza M1 57–68)
that lacks biological activity with the A6-TCR is shown as a reference
(
). C, Tax8 does not antagonize A6-bearing T-cells
for IFN-