From the Ludwig Institute for Cancer Research,
Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland,
the ¶ Department of Immunology, INSERM U277, Pasteur Institute,
75015 Paris, France,
Centre d'Immunologie, INSERM/CNRS, 13288 Marseille-Luminy, France, § Institut Le Bel, Louis Pasteur
University, 67404 Strasbourg, France, and ** Centre for Biotechnology,
Department of Chemistry EPFL, 1015 Lausanne, Switzerland
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ABSTRACT |
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To elucidate the structural basis of T cell
recognition of hapten-modified antigenic peptides, we studied the
interaction of the T1 T cell antigen receptor (TCR) with its ligand,
the H-2Kd-bound Plasmodium berghei
circumsporozoite peptide 252-260 (SYIPSAEKI) containing photoreactive
4-azidobenzoic acid (ABA) on P. berghei circumsporozoite
Lys259. The photoaffinity-labeled TCR residue(s) were
mapped as Tyr48 and/or Tyr50 of complementary
determining region 2 CD8+ cytotoxic T lymphocytes
(CTL)1 recognize by means of
their T cell antigen receptor (TCR) antigenic peptides, usually 8-10 amino acids long, bound to major histocompatibility complex (MHC) class
I molecules on target cells (1-4). However, CD8+ (and
CD4+) T cells can also recognize antigenic peptides
containing nonpeptidic moieties, such as carbohydrates or haptens, like
trinitrophenyl, azobenzenearsonate, fluorescein, or phenylazides
(5-15). Such T cells can be readily elicited and play a role in
diseases, e.g. allergies, contact dermatitis, and eczema
(16). The recognition of modified peptides is highly specific, and even
small changes in the hapten or carbohydrate moiety can dramatically
affect antigen recognition (13-18). This is reminiscent of
immunoglobulins, which can be raised against and specifically bind such
structures (19, 20). While x-ray crystallographic studies have revealed
how antibodies bind haptens, little is known about how TCR do this. This is of particular interest, because TCR genes, unlike
immunoglobulin genes, have no somatic mutations allowing affinity
maturation. Moreover, TCR need to recognize hapten or carbohydrate
moieties in the context of an MHC-peptide complex in a predefined
orientation (21-25).
Available three-dimensional structures of TCR-ligand complexes revealed
a consensus "diagonal" TCR-ligand orientation, in which the
MHC-bound peptide runs diagonally between the CDR3 loops, extending
from CDR1 Hapten or carbohydrates conjugated with antigenic peptides are part of
the epitope recognized by TCR (5, 8-15). TCR specific for
hapten-modified antigenic peptides typically exhibit preferential usage
of certain V We have previously generated and characterized two families of
H-2Kd-restricted CTL clones, specific for two different
photoreactive derivatives of the Plasmodium berghei
circumsporozoite peptide PbCS-(252-260) (SYIPSAEKI) (13, 15). In one
peptide derivative, ABA was conjugated with PbCS Lys259,
whereas P-255 was replaced by Lys(ABA) in the other. In addition, to
prevent Kd-peptide derivative complex dissociation, PbCS
Ser252 was replaced with iodo-4-azidosalicylic acid (IASA),
which upon selective photoactivation permitted covalent attachment of
the peptide derivative to Kd (26). The ABA, but not the
IASA group, was part of the epitope recognized by these CTL. The two
families of CTL clones were non-cross-reactive, and exhibited different
TCR sequences (13, 15).
In this study, we describe the interaction of the TCR of the T1 CTL
clones with its ligand, Kd-bound IASA-YIPSAEK(ABA)I. Using
mutational analysis, mapping of the photoaffinity-labeled site(s) and
molecular modeling, we identified a specific binding mode, how the T1
TCR binds the ABA group. We propose that this binding principle has
universal aspects.
Peptide and Peptide Derivative Synthesis--
Amino acids and
other chemicals were obtained from Bachem Finechemicals AG (Bubendorf,
Switzerland), Sigma Chemie (Buchs, Switzerland), and Neosystems
(Strasbourg, France). Synthesis and characterization of peptide
derivatives was performed as described previously (15, 17, 26).
HPLC-purified peptide derivatives were reconstituted in PBS at 1 mM. The specific radioactivity of 125I
conjugates was approximately 2000 Ci/mmol.
Cellular Assays--
For the cytolytic assay,
51Cr-labeled P815 cells (5 × 103
cells/well) were incubated for 1 h at 37 °C in medium
containing 10-fold dilutions of peptide derivatives, followed by UV
irradiation at Kd and TCR Photoaffinity Labeling--
All
photoaffinity labeling procedures were performed as described
previously (15, 17). In brief, for TCR photoaffinity labeling,
107 cpm of Kd-125IASA-YIPSAEK(ABA)I
were incubated with 107 T1 CTL on ice for 3 h,
followed by UV irradiation at 312 ± 40 nm. For peptide mapping,
T1 CTL (4 × 107) were incubated likewise with
Kd-SYIPSAEK(125IASA)I (1 mCi) in 2 ml of medium
containing Soluble T1 TCR and Mutants--
T1 Soluble Kd and Mutants of Soluble
Kd--
A full-length Kd cDNA cloned in
pKC expression vector (promotor SV40, Hanahan) was double-digested with
HindIII and XbaI in order to excise the sequences
encoding the cytoplasmic and transmembrane domains up to nucleotide
966. The vector was religated using a HindIII-XbaI linker containing a stop codon,
giving rise to translated C terminus RWKLA-stop. This Kd
cDNA was cloned into the pcDNA3 expression vector (Invitrogen) under control of the cytomegalovirus promoter. Site-directed
mutagenesis was performed using the TransformerTM kit
(CLONTECH) according to the manufacturer's
instructions. Full-length cDNA coding for Peptide Mapping--
All procedures have been described
previously (13, 15). Enzymes were obtained from Boehringer Mannheim
(Rotkreuz, Switzerland) and used as recommended (31). In brief,
photoaffinity-labeled T1 TCR was reduced, alkylated, and reconstituted
in 500 µl of 100 mM Tris, pH 8.0 (for tryptic digests) or
100 mM phosphate buffer, pH 7.8 (for V8 digests) containing
10% acetonitrile. Aliquots of enzymes (10 µg) were added in 12-h
intervals, and after 48 h of incubation at 37 °C, the digests
were subjected to reverse phase HPLC on an analytical C-18 column
(4 × 250 mm, 5-µm particle size, Vydac, Hisperia, CA). The
column was eluted with a linear gradient of acetonitrile in 0.1%
trifluoroacetic acid, rising within 1 h from 0 to 75%. Elution of
radioactivity was monitored by Molecular Modeling--
A homology model of the T1 TCR and the
Kd-SYIPSAEK(ABA)I complex was built using the MODELLER
program (33) based on the crystal coordinates of TCR A6 (V Effect of PbCS Peptide Derivative Mutations on T1 TCR-Ligand
Binding and Antigen Recognition by T1 CTL--
To obtain information
on PbCS(ABA) contacts with T1 TCR, several PbCS(ABA) variants were
assessed by TCR photoaffinity labeling with soluble
Kd-125IASA-YIPSAEK(ABA)I complexes (Fig.
1A). The replacement of PbCS Pro255 by Ala, Asp, or Ser reduced T1 TCR labeling by 10-, 100-, and 17-fold, respectively, whereas replacement by Leu increased
it 2-fold, suggesting that voluminous aliphatic residues in this position stabilize, and polar ones destabilize, T1 TCR-ligand binding.
Alanine substitution of PbCS Ser256 impaired T1 TCR
photoaffinity labeling by 95%. Substitution of PbCS Glu258
with alanine or glutamine obliterated detectable T1 TCR labeling, indicating that Glu258 forms a polar contact with T1 TCR.
Shortening of PbCS Lys259 by one methylene group
(YIPSAEOrn(ABA)I) also abolished T1 TCR labeling, indicating that the
full spacer length was required.
To define the interaction of ABA with T1 TCR, PbCS(ABA) variants with
modified ABA were examined. These nonphotoreactive compounds were
assessed in a cytolytic assay as derivatives of SYIPSAEK(ABA)I (Fig.
1B). Cloned T1 CTL killed target cells sensitized with
SYIPSAEK(benzoic acid)I approximately 100-fold less efficiently than
those sensitized with SYIPSAEK(ABA)I. Replacement of the
phenylazide by a methyl group (SYIPSAEK(Ac)I) obliterated detectable
antigen recognition, while introduction of an iodine and hydroxy
substituent in ABA (YIPSAEK(IASA)I) reduced the efficiency
of antigen recognition by 8-fold. These results indicate that the
phenylazide of the ABA moiety was essential for antigen recognition and
that changes of substituents predictably affected the efficiency of recognition.
Effect of Kd Mutations on T1 TCR Photoaffinity
Labeling--
To identify Kd-TCR contacts, we prepared
soluble Kd and 12 Kd mutants containing single
alanine substitutions on the surface of the Effect of T1 TCR Mutations on T1 TCR Photoaffinity
Labeling--
To define ligand contact residues, soluble T1 TCR and 31 mutants were prepared and tested by T1 TCR photoaffinity labeling with
soluble Kd-125IASA-YIPSAEK(ABA)I (Fig.
3). Seven of these mutations reduced TCR
photoaffinity labeling by Localization of the Photoaffinity-labeled Site(s) on T1
TCR--
To localize the photoaffinity-labeled site(s), T1 TCR was
photoaffinity-labeled with SYIPSAEK(125IASA)I, a derivative
that was efficiently recognized by T1 CTL (Fig. 1B) and,
being monovalent, precluded cross-linking with Kd. Since T1
TCR was photoaffinity-labeled exclusively at the
To verify this, it was treated with CNBr, which cleaves at methionine
residues. This resulted in the formation of a new labeled fragment,
which eluted from the C18 column after 34-35 min and migrated on
SDS-PAGE with an apparent Mr of approximately
2500 Da (Fig. 4, lane 4). Since the variable
domain of the T1 TCR
To substantiate this, we tested whether the labeled digest fragment
resulting after digestion with V8 and Asp-N contained an arginine, by
extensively digesting it with Arg-C. This treatment resulted in a new
labeled fragment that according to HPLC and SDS-PAGE was
indistinguishable from the smallest fragment obtained after digestion
of the labeled V8 digest fragment with Arg-C (Fig. 4, lane
8). By contrast, no change was observed when protease Lys-C
was used (data not shown). Together, these findings demonstrate that
the photoaffinity-labeled site(s) was contained in the uncharged sequence 45-56 (LIHYSYGAGSTE).
T1 TCR Photoaffinity Labeling Involved Tyrosine
Residues--
Because this sequence contains two tyrosines and ABA and
IASA have low, but measurable affinity for tyrosine (14, 15), we
examined whether the T1 TCR photoaffinity labeling involved tyrosine.
To this end, T1 TCR was biosynthetically labeled with [3H]tyrosine and photoaffinity-labeled with
Kd-SYIPSAEK(125IASA)I. Following digestion
with V8 and Asp-N, HPLC showed the same 125I profile as
observed previously and various 3H-labeled materials (Fig.
6A). The
125I-labeled material was extensively digested with
cathepsin C and carboxypeptidase P. HPLC showed that the major
125I-labeled material also contained 3H (Fig.
6B). On SDS-PAGE, this material migrated at the gel front, i.e. had a Mr of less than
1000-1200, confirming extensive, probably complete degradation (data
not shown). Tyrosine and monoiodotyrosine eluted from the HPLC column
in fractions 6, 7, and 19, respectively, i.e. both earlier
than the double-labeled digest fragment (Fig. 6C). These
results indicate that the T1 TCR Model of the T1 TCR-Kd-SYIPSAEK(ABA)I Complex--
To
evaluate our data in structural terms, we built a model of the T1
TCR-Kd-SYIPSAEK(ABA)I complex. Accurate homology modeling
of the T1 TCR and Kd was made possible by the availability
of crystal coordinates of closely related TCR and Kb (TCR
2C and TCR 14.3 express V
The phenyl rings of
The Kd-bound peptide runs diagonally between the CDR3
loops, and its termini are located underneath the CDR1 loops (Fig.
7A). According to the model, residues Kd
Glu58, Glu62,
Gln72, Gln149,
Asp152, Glu154, Ala158,
Tyr155, Tyr159, and
Glu166 form hydrogen bonds with T1 TCR (Table I). Alanine substitution of the underlined residues significantly reduced T1 TCR photoaffinity labeling (Fig. 2). The predicted contacts
with Kd Glu58 and Ala158 are in
accordance with TCR mutational analysis (Fig. 3). TCR-MHC contacts 1, 6, and 7 (Table I) were also observed in the A6 TCR-HLA-A2-Tax complex
(21), and the contacts 2-4 and 6 were observed in the 2C
TCR-Kb-dEV8 complex (23). According to the model, the PbCS
residues Pro255, Ser256, Glu258,
and Lys259(ABA) interact with T1 TCR (Table I). Indeed,
mutation of these residues, especially of Glu258 and
Lys259(ABA), substantially impaired T1 TCR photoaffinity
labeling or antigen recognition by T1 CTL (Fig. 1). For
Pro255, the model shows that the upper part of the nonpolar
proline ring makes a van der Waals contact with the methylene group of The present study uses peptide mapping, mutational analysis, and
molecular modeling to describe in structural terms how TCR can avidly
and specifically bind a hapten-modified peptide in the context of a MHC
class I molecule. The hapten, photoreactive ABA, conjugated at the
penultimate residue of the PbCS peptide SYIPSAEKI, constituted an
essential part of the epitope recognized by T1 CTL (Figs. 1 and 2). The
fact that peptide mapping allowed localization of the
photoaffinity-labeled site(s) on T1 TCR (Figs. 4 and 6) indicates that
this labeling was site-specific. In view of the high chemical
reactivity of the radicals produced by UV irradiation (14, 15) of
phenylazides and the flexible nature of the lysine side chain, this
implies that the ABA group associated with the T1 TCR in a well defined
orientation. If this were not so, heterogeneous photo-cross-linking
would occur to preclude reproducible and resolved peptide mapping
(40).
To better understand in structural terms the interaction of the
photoreactive ligand side chain with the T1 TCR, we modeled the T1
TCR-Kd-SIPSAEK(ABA)I complex. While available crystal
coordinates of related TCR and MHC class I molecules permitted homology
modeling of most of the system with good accuracy, the main problems
concerned the docking the TCR to the ligand and the positioning of
certain CDR loops. The docking was based on the diagonal orientation
observed for all TCR-ligand complexes whose three-dimensional
structures have been elucidated (21, 23-25). This orientation involves
conserved TCR-MHC class I contacts, mainly between TCR V According to the model, ABA inserts between the side chains of
While our model is in good agreement with most of the mutational data,
it failed to account for the effects of three TCR mutations ( Interestingly, the majority of CTL clones obtained from mice immunized
with IASA-YIPSAEK(ABA)I expressed V Although the interaction of ABA with T1 TCR constituted an important
part of T1 TCR-ligand binding, free peptide derivative, even at high
concentration (10 µM), was unable to photoaffinity-label T1 TCR.3 However, it has been reported that in other
systems hapten in polymeric form or hapten-conjugated peptides at high
concentrations can directly bind to TCR, i.e. that some
hapten-reactive T cells may have promiscuous MHC restriction (7, 10).
This is reminiscent of antibodies that can strongly bind small organic
molecules. As shown by x-ray crystallography, such antibodies utilize
residues from different CDR loops (19, 20). Comparison of the
three-dimensional structures of the complexes of fluorescein with a
monoclonal anti-fluorescein antibody and a fluorescein-specific TCR
showed that both molecules use very similar principles in binding
hapten (19, 41). Although TCR specific for hapten-modified peptides are
unlikely to achieve the high affinities by which antibodies can bind
hapten, they may reach higher affinities than TCR specific for
conventional peptides, since cells expressing such high affinity TCR
are prone to be eliminated by negative selection.
The present study suggests that TCR are able to bind specifically
hapten-conjugated peptides by selection of particular V We and others have previously observed that CTL can also be readily
elicited and specifically recognize haptens conjugated in position 4 or
5 of the peptide (11, 13). In a previous study, we mapped the
photoaffinity labeled site(s) for a TCR specific for IASA-YIK(ABA)SAEKI
(13). The labeled site(s) was located in the V These results provide significant insights into the structural basis of
T cell recognition of hapten-conjugated MHC binding peptides. TCR
expressing selected V (CDR2
). Other TCR-ligand contacts were
identified by mutational analysis. Molecular modeling, based on
crystallographic coordinates of closely related TCR and major
histocompatibility complex I molecules, indicated that ABA binds
strongly and specifically in a cavity between CDR3
and CDR2
. We
conclude that TCR expressing selective V
and CDR3
sequences form
a binding domain between CDR3
and CDR2
that can accommodate
nonpeptidic moieties conjugated at the C-terminal portion of peptides
binding to major histocompatibility complex (MHC) encoded proteins.
INTRODUCTION
Top
Abstract
Introduction
References
to CDR1
(21, 23-25). In this orientation, the CDR3
loops can interact extensively with peptide side chains, which are
mainly located in the center of MHC molecules, as well as with residues
of the MHC
-helices. The
-helices of MHC class I molecules are
elevated at the N-terminal portions; therefore, the approximately
planar surface of the TCR ligand binding site can realize the best
contact with the ligand in a diagonal orientation (21).
/J
, and/or specific junctional sequences (13, 15,
18). We used as hapten photoreactive 4-azidobenzoic acid (ABA). This
allowed assessment of TCR-ligand binding by TCR photoaffinity labeling
and identification of the photoaffinity-labeled site(s), i.e. the contact(s) of the hapten with the TCR (13, 15).
EXPERIMENTAL PROCEDURES
350 nm. Cloned T1 CTL (1.5 × 104
cells/well) were added, and after 4 h of incubation at 37 °C, released 51Cr was determined. The specific lysis was
calculated as 100 × ((experimental
spontaneous
release)/(total
spontaneous release)). The relative antigenic
activities were calculated by dividing the concentration of
IASA-YIPSAEK(ABA)I required for half-maximal lysis by that required for
the variant peptide derivatives. These values were normalized by
division with the corresponding relative Kd competitor
activities (14, 15, 17).
2-microglobulin (2.5 µg/ml). After UV
irradiation at
350 nm, cells were washed twice and lysed in
phosphate-buffered saline (1 × 107 cells/ml)
containing 0.7% Nonidet P-40, HEPES, phenylmethylsulfonyl fluoride,
leupeptin, and iodoacetamide. The detergent-soluble fractions were
subjected to immunoprecipitation with anti-TCR C
monoclonal antibody
H57-597. The immunoprecipitates were analyzed by SDS-PAGE (10%,
reducing conditions) and quantified using a PhosphorImager and the
ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). S.D.
values were calculated from 2-4 experiments.
and
cDNAs
extending from the 5' terminus up to, but not including, bases encoding
the extracellular membrane-proximal cysteine residues were generated by
reverse transcription on total T1 CTL RNA followed by polymerase chain
reaction (PCR) amplification. The DNA fragments encoding a linker
sequence and leucine zipper (LZ) components were generated by using
oligonucleotides and PCR on templates pACID and pBASE (27). The T1
TCR-leucine zipper cDNAs were prepared by using recombinant PCR on
these templates. The T1
LZ and T1
LZ cDNA containing basic and
acidic LZ, respectively, were cloned into pCR-script (Stratagene) and
subcloned into the EcoRI site of the mammalian expression
vector pCI-neo (Promega). All PCR amplifications were performed using
Pfu DNA polymerase (Stratagene), and both strands of cloned
inserts were sequenced and found to be error-free. TCR mutants were
generated using the QuickChange site-directed mutagenesis kit
(Stratagene) following the suppliers instructions. 293T cells (ATCC)
were transfected with pT1
LZ and pT1
LZ DNA (1:2 ratio) for
transient expression of soluble
T1 TCR following published
procedures (28). After 2 days, supernatants were harvested, and T1 TCR
concentrations were equalized. Preparation of T1 single chain Fv
cDNA constructs and protein expression were performed as described
previously (29).
2m was prepared and
inserted in the same vector using the proper linkers (30).
-counting of 1-ml fractions. For
destructive digestion the double-labeled V8 and Asp-N digest fragment
was reconstituted in 300 µl of citrate buffer (50 mM, pH
5.5) containing 50 mM NaCl and Nonidet P-40 (0.01%) and
incubated at 37 °C for 36 h with cathepsin C and
carboxypeptidases P. Enzymes (5 µg) were added every 12 h. For
biosynthetic labeling of T1 TCR with [3H]tyrosine, CD8
/
-transfected T1.4 T cell hybridomas (0.7 × 106) were incubated in tyrosine-deficient Dulbecco's
modified Eagle's medium supplemented with fetal calf serum (5%) and 5 mCi of [3H]tyrosine (NEN Life Science Products; specific
activity of 142 Ci/mmol) at 37 °C for 20 h. The washed cells
were photoaffinity-labeled with
Kd-SYIPSAEK(125IASA)I (specific radioactivity
of 20 Ci/mmol). The Mr values of the labeled
digest fragments were assessed by SDS-PAGE as described (32).
2.3,
J
24; V
12.3, J
2.1)-HLA-A2-Tax peptide complex (21), TCR 2C
(V
3, J
58; V
8.2, J
2.4) (22, 23), TCR 14.3
-chain
(V
8.2, J
2.1) (34), TCR 1934.4 V
(V
4.2) (35), and
H2-Kb (3). The related sequences of corresponding chains
were aligned using a dynamic programming method implemented in the
MODELLER program (33). An all atom model of the complex was built using MODELLER by satisfaction of spatial restraints obtained from the alignment and parameters in the program. A distance restraint was
introduced initially between the phenyl rings of ABA and
-Tyr48 and
-Tyr50, respectively. Side
chain orientations were optimized using a backbone-dependent rotamer library (36, 37). CDR1
and
CDR2
loops were not subsequently refined, since their conformation was modeled from the TCR 2C, which has the same V
8.2 as TCR T1. For
the other CDR loops, the conformations with the low energies were
identified by simulated annealing with the rest of the structure fixed.
From these, the final loop orientations were selected by using data
from the mutation experiments. The resulting structure was refined with
500 steps of steepest descent energy minimization using the CHARMM
(version 25) program (38) with the all-atom PARAM 22 parameter set
(39). No significant violation of spatial restraints was found for
-Tyr48,
-Tyr50, and K(ABA)
after optimization, indicating that the imposed distance restraint does
not imply a distortion of the structure. Details concerning the
modeling will be presented
separately.2
RESULTS
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Fig. 1.
Effect of PbCS peptide derivative mutations
on T1 TCR photoaffinity labeling and antigen recognition by T1
CTL. A, the indicated radiolabeled peptide derivatives
were photo-cross-linked to soluble Kd, and the resulting
covalent Kd-peptide derivative complexes were used for TCR
photoaffinity labeling on cloned T1 CTL. The labeling observed for
Kd-125IASA-YIPSAEK(ABA)I was defined as 1, and
labeling values for the ligand variants are expressed relative to this
value. B, alternatively, the specific lysis of
51Cr-labeled P815 cells was assessed in the presence of the
indicated PbCS peptide derivatives. The antigenic activities are
expressed relative to SYIPSAEK(ABA)I and normalized with the relative
Kd competitor activities; thus, by definition the
normalized relative antigenic activity of SYIPSAEK(ABA)I was 1 (see
"Experimental Procedures"). Each experiment was performed in
triplicate and repeated at least once. The mean values and S.D. values
were calculated from all experiments.
1 or
2 helices (Fig.
2). After photo-cross-linking with radiolabeled 125IASA-YIPSAEK(ABA)I, TCR-ligand binding was
assessed by T1 TCR photoaffinity labeling, as described above. Six of
the Kd mutations impaired T1 TCR-ligand binding by
50%.
Two were on Kd
1 (E62A and Q72A), and four were on
Kd
2 (Q149A, D152A, Y155A, and E166A). Some
Kd mutations increased T1 TCR photoaffinity labeling by up
to 20% (S69A, R79A, and E163A).
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Fig. 2.
Effect of Kd mutations on T1 TCR
photoaffinity labeling. Soluble Kd molecules
containing single alanine substitutions in the indicated position were
photo-cross-linked with 125IASA-YIPSAEK(ABA)I, and their
ability to photoaffinity-label T1 TCR was assessed as described for
Fig. 2. Each experiment was performed 2-4 times.
90%. Three of these were in CDR3 loops,
three others were in CDR1, and one was in CDR2
. In addition, six
other mutations reduced T1 TCR photoaffinity labeling by
50%. Four
of these were in CDR2
, one was in CDR2
, and one was in CDR3
.
Several mutations increased TCR photoaffinity labeling by up to 60%
(e.g.
N27A,
Y50F, and
T55A). Mutants labeled with an asterisk were T1 TCR Fv single chain constructs (29).
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Fig. 3.
Effect of T1 TCR mutations on TCR
photoaffinity labeling. Soluble T1 TCR comprising the variable and
constant domains of both chains and mutants containing single alanine
substitutions in the indicated positions were incubated with soluble
covalent Kd-125IASA-YIPSAEK(ABA)I complexes at
0-4 °C for 30 min. After UV irradiation, TCR photoaffinity labeling
was assessed as described for Fig. 2. The mutants labeled with an
asterisk were single chain Fv T1 TCR constructs. The mean
values and S.D. values were calculated from two experiments.
-chain (15), the
photoaffinity-labeled TCR was directly subjected to peptide mapping.
After extensive digestion with trypsin, the resulting digest fragments
were separated by reverse phase HPLC. The major labeled digest fragment
eluted from the C-18 column after 30 min and according to SDS-PAGE was
homogenous and had an apparent Mr of
approximately 3000 (Fig. 4,
lane 2). When protease V8 was used instead of
trypsin, a major labeled material eluted from the column after 32-33
min, which migrated on SDS-PAGE with an apparent
Mr of approximately 8300 (Fig. 4,
lane 1). The size of this fragment was bigger
than any theoretical V8 fragment of the variable domain of the T1 TCR
-chain (Fig. 5B),
suggesting that protease V8, even after extensive digestion, omitted a
cleavage site. Since this enzyme primarily cleaves C-terminal to Glu
and hence may fail to cleave after Asp (31), the labeled V8 digest
product was digested with protease Asp-N. Essentially the same HPLC
profile was observed; however, on SDS-PAGE this material migrated with an apparent Mr of approximately 2000 (Fig. 4,
lane 3), indicating that V8 failed to cleave at an aspartic acid. This
residue probably was
-Asp38, because the big reduction
in size (approximately 6300 Da) may correspond to the size of the
segment 2-37, which contains the glycosylation site
-Asn24 (Fig. 5B and Ref. 34). Accordingly,
the labeled V-8 digest fragment would be 2-56 (Fig.
5B).
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Fig. 4.
Localization of photoaffinity-labeled site(s)
on T1 TCR -chain. T1 TCR was
photoaffinity labeled with soluble
Kd-SYIPSAEK(125IASA)I and digested with
different proteases. The resulting fragments were separated by C-18
reverse phase HPLC, and the 125I-containing materials were
analyzed by SDS-PAGE. Lane 1, V8 digest;
lane 2, tryptic digest; lane
3, digest with V8 and Asp-N; lane 4,
digest with V8 and CNBr cleavage; lanes 5,
6, and 7, HPLC fractions 31, 30, and 29, respectively, of V8 and Arg-C digest; lane 8,
digest with V8, Asp-N, and Arg-C. Representative experiments are shown.
Each experiment was repeated 1-5 times.
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Fig. 5.
Amino acid sequence of T1 TCR
- and
-chain. For the
-chain, which contains the labeled site(s), the cleavage sites for
protease V8 (E and D) and for trypsin
(R and K) are marked. Cysteines and methionines
are printed in boldface type. The digest fragment
45-56 containing the photoaffinity-labeled site(s) is shown in
gray. For both TCR chains, the CDR sequences are shown in
boxes, and the residues subjected to mutational analysis are
marked by triangles; open triangles
indicate residues sensitive to mutation. The constant domains on both
TCR chains start at position 112.
-chain contains only one methionine, this
confirmed that the labeled V-8 digest fragment consisted of residues
2-56 (Fig. 5B). The observed shift in
Mr suggested that the labeled site(s) was
located C-terminal to
-Met32, i.e. in the
segment 33-56. Moreover, the predicted labeled V8 digest fragment
contains three arginine residues (Arg9, Arg36,
and Arg44). To assess their presence, it was partially
digested (6 h) with Arg-C. As assessed by HPLC, this treatment produced
earlier eluting radioactive materials, probably of mixed composition,
as suggested by the broad range of elution (29-31 min). SDS-PAGE
analysis of fractions 29-31 showed three new labeled species,
migrating with apparent Mr of approximately
1800, 2600, and 7000, respectively, with the smaller ones eluting
earlier from the HPLC column (Fig. 4, lanes
5-7). Upon extensive digestion with Arg-C, only the
smallest fragment was observed (data not shown). These findings confirm that the labeled primary V8 digest fragment was residues 2-56 and
suggest that the labeled site was contained in the segment 45-56.
-chain was photoaffinity-labeled at
tyrosine residue(s) and demonstrate that this tyrosine modification was
not a UV irradiation mediated trans-iodination.
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Fig. 6.
T1 TCR is photoaffinity-labeled at tyrosine
residue(s). A, soluble T1 TCR, biosynthetically labeled
with [3H]tyrosine, was photoaffinity-labeled with soluble
Kd-SYIPSAEK(125IASA)I. Following extensive
digestion with protease V8 and Asp-N, the digest fragments were
separated by C-18 reverse phase HPLC, and 0.75-ml fractions were
counted for 125I (diamonds) and 3H
(squares). B, the major 125I-labeled
material (fractions 42-44) was treated with cathepsin C and
carboxypeptidase P, and the resulting fragments were analyzed likewise.
The 125I spill-over was subtracted from the 3H
cpm. C, as references, tyrosine (squares) and
iodotyrosine (diamonds) were subjected to HPLC, and the OD
was measured at 275 nm.
8.2 as TCR T1 and TCR 1934.4 expresses a
V
of the same subfamily). The C
root mean square deviation between our model and (i) the V
of the 2C TCR was 0.35 Å, (ii) the
V
of the 1934.4 TCR was 1.68 Å, (iii) the variable domain of
Kb was 0.53 Å, and (iv) the A6 TCR-HLA-A2-Tax peptide
complexes was 1.83 Å (CDR3 included). The diagonal orientation of the
T1 TCR relative to the Kd-PbCS(ABA) complex and the
positioning of the CDR loops resulted in a pocket between CDR1
,
CDR2
, and CDR3
. The bottom of the pocket is formed by
-Arg99 and
-Glu56, one side by CDR3
residues Asn96 and Asn97 and the other side by
CDR2
residues Tyr48, Tyr50, and
Asn31 of CDR1
(Fig. 7).
The Lys259(ABA) side chain was orientated such that it
inserts into this cavity between the side chains of
-Tyr48 and
-Tyr50. The first two
methylene groups of K259(ABA) have van der Waals contacts with
Kd Trp73, and the ABA carbonyl forms a hydrogen
bond with its indol nitrogen. These interactions restrict the mobility
of the K(ABA) side chain and keep it in a slanted orientation.
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Fig. 7.
Molecular modeling of the T1
TCR-Kd-SYIPSAEK(ABA)I complex. A, top view
of the surface of the T1 TCR ligand binding site with bound
SYIPSAEK(ABA)I in the bound state ( -chain on the left and
-chain on the right). Peptide residues are shown in
yellow, surface-exposed acidic TCR residues are shown in
red, and basic ones are shown in blue. The C
backbones of the underlying CDR1 loops are shown in light
yellow, those of the CDR2 loops are shown in
light green, and those of the CDR3 loops are
shown in purple. B, stereo view of K259(ABA)
(green) and contacting T1 TCR residues. CDR3
residues are shown in
light blue, CDR2
residues are shown in
dark yellow, CDR1
and CDR3
residues are
shown in white, and Kd Trp73 is
shown in light yellow. The dotted
lines indicate hydrogen bonds. The images were
produced with the MOLMOL program (42).
-Tyr48,
-Tyr50, and
ABA are nearly parallel and equally spaced at a distance of
approximately 3.0 Å. This is in agreement with the finding that
-Tyr48 and/or
-Tyr50 were
photoaffinity-labeled sites (Figs. 4 and 6). Five other TCR residues,
-Asn96,
-Asn97,
-Arg99,
-Asn31, and
-Glu95, also interact with
the ABA moiety. The
-amide nitrogen (ND2) of
-Asn96
forms a hydrogen bond to the ABA carbonyl OF (see Table
I for atom labels); the C
of
-Asn97 makes a van der Waals contact with its CE2; OD1
of
-Asn31 makes a polar interaction of the C-H ...
X type with its CD1 and CE1 and OE1 of
-Glu95
with its NZ (Table I). There is also a weak hydrogen bond between the
OH group of
-Tyr50 and ABA CE2 as well as a polar
contact between the OH group of
-Tyr48 and N2 of ABA.
Finally, there is a hydrogen bond between NH1 and NH2 of
-Arg99 and N3 of ABA. These interactions are
consistent with the data from the mutational analysis (Figs. 1-3),
although there was no quantitative evaluation of the free energies
involved.
T1 TCR-ligand contacts predicted by model
-Gly95 and a hydrogen bond with O of
-Arg94, which is consistent with the observation that
replacement of Pro255 with Ala, Ser, Asn, or Asp decreased
T1 TCR photoaffinity labeling (Fig. 1). For PbCS Ser256,
the model predicts a hydrogen bond between its OH group and OD1 of
N96, and for PbCS Glu258 contacts with
-Gln97 and Kd Lys146. Alanine
substitution of
-Gln97 dramatically reduced T1 TCR
photoaffinity labeling (Fig. 3) and of Kd
Lys146 peptide binding to Kd (data not shown),
since this residue also interacts with the C-terminal carboxyl group of
the peptide (3, 4).
DISCUSSION
and
residues of the MHC
1 and
2 helices (21, 23, 24). Our model is in accordance with these contacts. Moreover, we made use of the
interaction of ABA with
-Tyr48 and
-Tyr50, as indicated by the observations that (i) the T1
TCR was photoaffinity-labeled at tyrosines of the CDR2
segment
45-56 (LIHYSYGAGSTE) (Figs. 4 and 6); (ii) TCR of the same
specificity, which expressed
-Tyr48, but not
-Tyr50, were photoaffinity-labeled also at the
-chain
(15); (iii) ABA and IASA have low affinities for Tyr and Trp (14); and
(iv) TCR-ligand binding, as assessed by inhibition of T1 TCR
photoaffinity labeling by the soluble Fv T1 TCR, correlated with TCR
photoaffinity labeling, except for the mutants
Y48F and
Y50F.3
-Tyr48 and
-Tyr50, which are part of a
pocket between CDR2
and CDR3
(Fig. 7). This position is
energetically favorable and provides extensive
-
interactions and
hydrogen bond formation (Table I). The ABA group was placed initially
with a constraining potential between these tyrosine side chains, but
it moved very little in subsequent energy minimization. The model shows
that nine contacts, involving seven different TCR residues, further
stabilize the interaction of the Lys(ABA) side chain with T1 TCR (Table
I). This is a consequence of a specific three-dimensional arrangement
of CDR3
, CDR3
, CDR2
, and CDR1
residues around the Lys(ABA)
side chain. Mutation of these TCR residues, as well as modification of
the Lys(ABA) side chain significantly affected T1 TCR photoaffinity
labeling and antigen recognition by T1 CTL (Figs. 1 and 3). Similarly,
TCR specific for peptides conjugated with benzoarsonate or fluorescein also utilize residues from different CDR loops for hapten binding (6,
41).
V49A,
T50A, and
E56A) (Fig. 3 and Table I). Possible explanations include the following. (i) Certain mutations may result in
conformational changes of CDR loops (e.g. the side chain of
-Val49 is in the center of CDR2
and forms various
contacts with the peptide backbone; therefore, the
V49A mutation
probably affects the CDR2
loop conformation). (ii) Some mutations
may affect intramolecular amino acid interactions, resulting in
reorientation of side chains engaged in TCR-ligand contacts
(e.g.
-Glu56 forms a hydrogen bond with
-Arg99, which interacts with N3 of ABA; thus,
the
E56A mutation may be explained by reorientation of the
-Arg99 side chain). (iii) Water was not included in the
model, which may account for some of these divergences.
1- and
J
TA28-encoded TCR, which were photoaffinity-labeled at both chains
(15). For one of them, the photoaffinity labeled sites were identified
as J
TA28-encoded tryptophan 97 and a residue of the V
1 segment 46-51, which contains
-Tyr48, but not
-Tyr50 (15). It thus appears that for these TCR, the ABA
group was inserted between
-Trp97 (CDR3
) and
-Trp48 (CDR2
), rather than two V
-encoded tyrosines.
and CDR3
sequences. From the data and the model, V
-encoded residues of CDR1
and mainly CDR2 play a key role in binding the Lys(ABA) side chain;
however, residues of CDR3
and CDR3
spatially and electronically
complement these interactions to an integral and sophisticated binding
mode (Fig. 7, Table I). We suggest that this binding principle has
universal aspects, i.e. that TCR expressing selected V
and CDR3
sequences are able to specifically bind haptens conjugated
at the penultimate residue of MHC binding peptides by means of a pocket
between CDR2
and CDR3
. As far as is known, and presumably for
reasons of chemical reactivity, most "hapten-reactive" TCR
recognize antigenic peptides containing a hapten-conjugated lysine (5,
11, 12). The long and flexible nature of the lysine side chain enables
haptens to bind in this cavity, in the framework of the canonical
diagonal orientation of TCR-ligand binding.
-encoded C-strand
segment 33-39, and computer modeling suggested that the Lys(ABA) side
chain inserted into a pocket between the two CDR3 loops (13).
Three-dimensional structure analysis showed that TCR indeed have a
cavity between the CDR3 loops and that it can accommodate side chains
of MHC-bound peptides (21, 22, 24).
and CDR3
sequences can form cavities
between CDR loops that can accommodate peptide-conjugated haptens in a
highly specific manner (Fig. 7 and Refs. 13 and 15). The structural
variability required for this is based mainly on junctional diversity,
but V-encoded TCR sequences, in some cases even framework residues,
play a role as well. This is consistent with the observation that
hapten-reactive T cells typically express different TCR sequences than
those recognizing the parental epitope (13-15).
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ACKNOWLEDGEMENTS |
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We thank Drs. E. Bernasconi and M. Jordan for excellent technical assistance; Drs. D. Kuznetsov and V. Jongeneel for computational work; Drs. R. Stote, D. York, and X. Lopez for aid in determining force field parameters for the ABA group; and J. Muller and K. Rey for preparing the manuscript.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Ludwig Institute of
Cancer Research, Chemin des Boversses 155, 1066 Epalinges, Switzerland.
Tel.: 41 21 692 59 88; Fax: 41 21 653 44 74; E-mail: iluesche{at}eliot.unil.ch.
The abbreviations used are: CTL, cytotoxic T lymphocyte(s); ABA, 4-azidobenzoic acid; CDR, complementary determining region; MHC, major histocompatibility complex; TCR, T cell antigen receptor(s); PbCS, P. berghei circumsporozoite; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; LZ, leucine zipper; IASA, iodo-4-azidosalicylic acid.
2 O. Michielin and M. Karplus, manuscript in preparation.
3 B. Kessler, O. Michielin, C. L. Blanchard, I. Apostolou, C. Delarbre, G. Gachelin, C. Grégoire, B. Malissen, J.-C. Cerottini, F. Wurm, M. Karplus, and I. F. Luescher, unpublished data.
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REFERENCES |
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