(Received for publication, October 7, 1996)
From the Ludwig Institute for Cancer Research,
Lausanne Branch, University of Lausanne,
1066 Epalinges, Switzerland
To study the interaction of T cell receptor with
its ligand, a complex of a major histocompatibility complex molecule
and a peptide, we derived H-2Kd-restricted cytolytic T
lymphocyte clones from mice immunized with a Plasmodium
berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI)
derivative containing photoreactive
N-[4-azidobenzoyl] lysine in
place of Pro-255. This residue and Lys-259 were essential parts of the
epitope recognized by these clones. Most of the clones expressed
BV1S1A1 encoded
chains along with specific complementary
determining region (CDR) 3
regions but diverse
chain sequences.
Surprisingly, all T cell receptors were preferentially photoaffinity
labeled on the
chain. For a representative T cell receptor, the
photoaffinity labeled site was located in the V
C-strand. Computer
modeling suggested the presence of a hydrophobic pocket, which is
formed by parts of the V
/J
C-, F-, and G-strands and adjacent
CDR3
residues and structured to be able to avidly bind the
photoreactive ligand side chain. We previously found that a T cell
receptor specific for a PbCS peptide derivative containing this
photoreactive side chain in position 259 similarly used a hydrophobic
pocket located between the junctional CDR3 loops. We propose that this
nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.
CD8+ CTL1 recognize
peptides bound to MHC class I molecules on the surface of target
cells by means of their TCR (1). Crystallographical studies showed that
some peptide side chains intrude into allele specific pockets of the
MHC molecule, whereas others are surface exposed and may interact with
TCR (2, 3). Conversely, crystallographical studies on a BV8S2A1 encoded
TCR chain and an AV4S2 TCR
chain fragment confirmed that TCR
are folded in an Ig-like manner but also indicated significant
structural differences, such as different hinge structures and
interstrand hydrogen bond formation (4, 5). Little is still known on
how TCR bind their ligand (MHC-peptide complexes). Based on theoretical
considerations, it has been proposed that V-encoded complementary
regions CDR1 and CDR2 primarily interact with the MHC molecule, such
that the junctional CDR3 are located over the MHC peptide binding
groove (6). Support for this concept mainly derives from experiments
indicating that CDR3 loops interact with residues of MHC bound peptides
(7, 8). One study using immunization of single chain TCR transgenic
mice with peptide variants suggested that CDR3
interacted with an
N-terminal residue of an MHC class II bound peptide and CDR3
with a
C-terminal one (9). A more recent study, using a similar strategy,
showed that an N-terminal peptide residue interacted with CDR1
or
CDR2
, while a C-terminal one interacted with CDR3
, implicating a
diagonal orientation, e.g. about 30 °C rotated (10). In
contrast, analysis of a Kb-restricted OVA-specific TCR
indicated that the most C-terminal residue of this epitope interacted
with CDR3
(11).
While these studies are in accordance with the current concept of
TCR-ligand interactions (6), there are observations that are difficult
to explain this way. For example, MHC class II-restricted tetanus toxin
tt830-844 reactive T cells exhibited preferential BV2S1 usage but
lacked junctional sequence conservation on both chains (12).
Furthermore, an Ld-restricted arsonate-reactive CTL clone
using the same AV3S1 sequence as an I-Ad-restricted
arsonate-reactive one, but an unrelated CDR3 junction, also
recognized this epitope, suggesting that in this system, CDR3 sequences
may not be important (13, 14). Moreover, we found that a TCR specific
for the Plasmodium berghei circumsporozoite PbCS peptide
252-260 (SYIPSAEKI) conjugated with 4-azidobenzoic acid (ABA) at
Lys-259 bound the photoreactive ligand side chain with CDR3
and V
C
-strand/CDR2
residues (15).
In this previous study, we modified the H-2Kd
(Kd)-restricted PbCS 252-260 by replacing Ser-252 with
photoreactive iodo-4-azidosalicylic acid (IASA) and by conjugating the
TCR contact residue PbCS Lys-259 with ABA, to make IASA-YIPSAEK(ABA)I.
CD8+ CTL clones were derived from mice immunized with this
conjugate. These recognized IASA-YIPSAEK(ABA)I as well as YIPSAEK(ABA)I
in a Kd-restricted manner, but not IASA-YIPSAEKI or the
parental PbCS peptide. Selective photoactivation of the IASA group
allowed covalent attachment of the conjugate to Kd
molecules. Incubation of these complexes with cloned CTL and photoactivation of the ABA group resulted in TCR photoaffinity labeling
(15). Unlike PbCS-specific CTL, these clones preferentially expressed
BV1S1A1 encoded TCR chains that were paired with J
TA28 encoded
chains (15). Sequences encoded by these TCR gene elements formed a
hydrophobic pocket, which was structured and located such that it could
efficiently accommodate the photoreactive ligand side chain. In the
present study, we replaced PbCS Pro-255 with K(ABA) and produced
likewise peptide derivative-specific CTL clones. This residue was
chosen because it is surface exposed and critical for T cell
recognition (16). Here we report that these CTL clones were remarkably
different from the ones described previously in terms of antigen
recognition, TCR sequences, and binding of the photoreactive ligand
side chain. For a representative TCR, we show a novel binding principle
by which TCR can efficiently and specifically bind a chemically
modified epitope.
Reagents for chemical synthesis were obtained from Bachem Finechemicals (Bubendorf, Switzerland), Pierce, and Sigma. All synthetic and analytical procedures were performed essentially as described (17, 18). In brief, peptides were synthesized on an Applied Biosystem Instruments 431 peptide synthesizer (ABI, Foster City, CA) using Fmoc (N-(9-fluorenyl)methoxycarbonyl) for transient protection. The deprotected peptides and peptide derivatives were purified by HPLC on a C-18 column (1 × 25 cm, Macherey Nagel, Oensingen, CH) using a Waters 600E HPLC system equipped with an in-line 1000 S diode array UV photo-spectrometer (Applied Biosystem Instruments). The column was eluted by a linear gradient of acetonitrile on 0.1% trifluoroacetic acid in water, rising within 1 h from 0 to 75%. All compounds displayed the expected UV spectra (17, 19, 20). ANBA-YIKSAEKI displayed UV absorption maxima at 214 and 280 nm, and the UV absorption at 350 nm was approximately 7% of the one at 280 nm. The mass of all purified compounds were verified by mass spectrometry on an LDI 7000 mass spectrometer (Linear Scientific, Reno, CA). Radiosynthesis of 125IASA-YIK(ABA)SAEKI and Dap(125ISA)-YIK(ABA)SAEKI was performed by radioiodination of ASA-Y(PO3H2)IK(ABA)SAEKI and Dap(ISA)-Y(PO3H2)IK(ABA)SAEKI, respectively, followed by enzymatic dephosphorylation. The HPLC-purified products were lyophilized and immediately used for photoaffinity labeling experiments.
Kd and TCR Photoaffinity LabelingSoluble
monomeric Kd-peptide derivative complexes were prepared by
incubating purified Kd with
125IASA-YIK(ABA)SAEKI followed by UV irradiation at 350
nm and FPLC gel filtration as described by Luescher et
al.(15, 17). For TCR photoaffinity labeling, 107 cpm
of Kd-"125IASA"-YIK(ABA)SAEKI were
incubated with 107 CTL, resuspended in 1 ml of Dulbecco's
modified Eagle's medium supplemented with 2% fetal calf serum and 20 mM HEPES, on ice for 3 h followed by UV irradiation at
312 ± 40 nm. For two-dimensional gel analysis and peptide
mapping, peptide derivative-Kd cross-linking was prevented
by using the monofunctional PbCS derivative Dap(ISA)-YIK(ABA)SAEKI as
described previously (15). 1 mCi of
Dap(125ISA)-YIK(ABA)SAEKI was incubated with 50 µg of
Kd for 2 h at ambient temperature prior to addition of
4 × 107 cloned CTL in 2 ml of medium (see above)
containing
-2 microglobulin (2.5 µg/ml). After 2-3 h of
additional incubation at 0-4 °C, the incubations were UV irradiated
at 312 ± 40 nm.
The
photoaffinity labeled cells were washed twice with cold PBS and lysed
on ice in PBS (1 × 107 cells/ml) supplemented with
0.7% Nonidet P-40, HEPES, phenylmethylsulfonyl fluoride, leupeptin,
and iodoacetamide as described (17). After centrifugation (3 min at
15,000 × g), the detergent-soluble fractions were
membrane filtered and subjected to immunoprecipitation with the
anti-TCR C mAb H57-597 or the anti-Kd
3 mAb
SF1-1.1.1 as described (17). For two-dimensional gel analysis and
peptide mapping, TCR were immunoprecipitated with H57-597 mAb on
protein A-Sepharose as described (17, 19, 20). The washed and
lyophilized Sepharose was resuspended in sample buffer for IEF, and
after a 1-h incubation at 37 °C, the supernatants were subjected to
disc IEF followed by SDS-PAGE as described (21). All antibodies were
obtained from American Type Culture Collection (ATCC, Rockville,
MD).
The photoaffinity labeled and
immunoprecipitated W13 TCR was reduced and denatured in 200 µl of 100 mM Tris buffer, pH 8.0, supplemented with 8 M
urea, 100 mM 2-mercaptoethanol, and 1% Nonidet P-40, at
37 °C for 1 h. To the supernatant, 400 µl of 500 mM Tris, pH 8.0, containing 100 mM iodoacetic
acid were added. After incubation for 1 h at 20-24 °C, the
samples were dialyzed at 4 °C in 25-kDa membrane tubings
(Spectrapore) against 3 × 1 liter of distilled water. Following
lyophilization, aliquots were reconstituted in 500 µl of either 100 mM Tris, pH 8.0 (for tryptic digests), or 100 mM phosphate buffer, pH 7.8 (for V-8 digests), containing 1 M guanidine hydrochloride, both containing 10%
acetonitrile, and subjected to tryptic or protease V-8 peptide mapping
as described (15) except that a C-18 column was used for HPLC (4 × 250 mm, 5 µm particle size, Vydac, Hisperia, CA). In brief, 10 µg aliquots of trypsin or protease V-8 (Boehringer Mannheim) were
added in 12-h intervals, and after 48 h of incubation at 37 °C,
the digests were subjected to HPLC. Radioactivity was monitored by counting of 1 ml fractions and chromatography by measuring the
A214 nm of standards including
NE-(2,4-dinitrophenyl)Lys (elution time 29.1 min), approximately YIPSAEKI (23.6 min), MAVDPIGHLY (26.7 min),
ASNENMDAM (30.1 min), and oxidized insulin
chain (37.7 min)
(Sigma). The maximal variation tolerated was ±1 min.
For secondary peptide mapping, labeled digest fragments were
reconstituted in 300 µl of 100 mM phosphate buffer, pH
7.4, containing 50 µg of bovine
-globulin and 10% acetonitrile.
Endoprotease Pro-C (gift from Dr. J. Gagnon, Biologie Structurale,
C.E.A. and C.N.R.S., URA 1333, Grenoble, France) was added in 10-µg
aliquots in 12-h intervals, and after 36-48 h, the samples were
subjected to HPLC. SDS-PAGE analysis of labeled digest fragments was
performed as described (22).
Cloned
CTLs were obtained as described previously (15, 16). In brief,
(BALB/c × C57BL/6) F1 mice were immunized with ConA spleen blasts
expressing covalent Kd-"ANBA"-YIK(ABA)SAEKI complexes.
Cultures of peritoneal exudate lymphocytes were stimulated in
vitro over two 7-day intervals. Viable cells were cloned 7 days
after the second in vitro stimulation by
fluorescence-activated cell sorting of CD8+ cells into
96-well plates. The clones were stimulated weekly with 2 × 104 -irradiated P815 pulsed before hand with 1 µM of ANBA-YIK(ABA)SAEKI and 3 × 105
-irradiated BALB/c splenocytes. All rapidly expanding cultures were
tested as described below. The U, V, and W clones were derived from
three different mice.
The cytolytic activity of the CTL clones under study was assessed in a standard 51Cr release assay as described previously (16, 23). In brief, 51Cr-labeled P815 cells or Kd transfected L cells (L-Kd) (2 × 103 cells/well) were incubated with a constant number of CTLs (6 × 103 cells/well) and serial dilutions of peptide derivative or peptide derivative variants in V-bottom microplates. The L-Kd and L-Kd mutants have been described previously (24). After 4 h of incubation at 37 °C, the released 51Cr was measured, and the specific lysis was calculated in percent as 100 × (experimental-spontaneous release)/(total-spontaneous release). The relative antigenic activities were calculated by dividing the concentration of ANBA-YIK(ABA)SAEKI required for half-maximal lysis by that required for the peptide derivative variants. The Kd competitor activity, expressing the ability of a peptide derivative to bind to Kd, was assessed in a recognition-based competition assay as described (23). In brief, 51Cr-labeled P815 cells were incubated with three-fold dilutions of the test peptide derivatives prior to addition of the antigenic HLA-Cw3 peptide 170-179 (RYLKNGKETL) and Cw3-specific cloned CW3/1.1 CTL. After 4 h of incubation, the 51Cr release was measured. The Kd competitor activity of each compound was calculated relative to the one of ANBA-YIK(ABA)SAEKI, which was defined as 1. For the sake of comparison, the relative antigenic activities of the different peptide variants were normalized by dividing the relative antigenic activity by the corresponding relative Kd competitor activities.
TCR Sequence AnalysisTotal RNA extraction was performed
using the isothiocyanate acid-phenol method. cDNA synthesis was
carried out on total RNA with AMV reverse transcripts following the
supplier instructions (Boehringer Mannheim, Rotkreuz, Switzerland).
Screening of junctional regions of and
chains was performed by
PCR using primers as described in Ref. 25. Purified PCR products were
sequenced with Sequenase Version 2.0 kit (U. S. Biochemicals,
Cleaveland, OH) and
[35S]dATP following the supplier
instructions. For sequencing of the V
of W13 and U4, the primers
W135
(GACCGAATTCAGATGACACTAAAGATGGAC) and W133
(CAACGTGGATCCACAGGGAACGTCTGAACTGG) were used.
Models of the W13 TCR and
Kd-ANBA-YIK(ABA)SAEKI complex were built using the ICM
software (26). For the chain, we used as template the crystal
coordinates of a BV8S2A1 encoded
chain (4). A crude model of the
W13 V
chain was obtained using the ProMod package (27) and was
subjected to a standard energy minimization with X-PLOR and followed by
a regularization of the geometry with ICM (the RMSD between the initial
and the regularized structures was 0.49 Å for non-hydrogen atoms).
Similarly, the model of the W13 TCR V
chain was obtained by homology
modeling using the x-ray structure of AV4S2 (5) as a template. The
resulting V
model (RMSD between the template and the modeled
structure was 1.0 Å for 106 C
atoms) was associated with the V
model to form a V
-V
complex, starting with the geometry of an Ig
VH/VL domain. After minimization of the
complex, possible conformations of the CDR3 loops were searched using a
local deformation Monte-Carlo procedure. The
Kd-ANBA-YIK(ABA)SAEKI complex was modeled on the basis of
the x-ray structure of H-2Kb as described before (15). As
the PbCS peptide derivative contains non-canonical amino acids, namely
K(ABA) and ANBA, models for these residues were generated using the
molecular editor of the HyperChem package. Atom charges were calculated
using the CNDO/2 method. The files containing the resulting coordinates
and charges were imported and processed by ICM to produce ICM library
entries used in subsequent modeling. The structure of the trimolecular complex, consisting of W13 V
-V
docked to the
Kd-peptide complex, was obtained by several cycles of the
following procedures and guided by the experimental data derived from
the mapping of the photoaffinity labeled site and the effects of
mutations in Kd: 1) local deformation Monte-Carlo search of
an optimal conformation for V
-V
CDR loops within the field of the
Kd-peptide surface; 2) Monte-Carlo search of the optimal
position of the side chains of all residues of the interface between
TCR and ligand; 3) a constrained Brownian-like "walk" of the ligand around the TCR; and 4) exhaustive minimization of the whole structure. The resulting structure was checked visually as well as by calculation of the local electrostatic interactions in the K(ABA) binding pocket.
To produce a new family of "photoprobe"-specific CTL clones,
we prepared a photoreactive derivative of the PbCS peptide 252-260 by
replacing Pro-255 with K(ABA). To cross-link the peptide derivative to
Kd, Ser-252 was replaced with IASA to make
IASA-YIK(ABA)SAEKI. As assessed in a recognition-based competition
assay, this derivative bound to Kd nearly 100-fold less
efficiently than the parental PbCS peptide, but replacement of IASA
with ANBA, N-IASA-2,3-L-diaminopropionic acid
(Dap(IASA)), or
N
-(iodo-salicyloyl)-2,3-L-diaminopropionic acid
(Dap(ISA)) restored efficient Kd binding (2-3-fold less
than the PbCS peptide). However, since ANBA-YIK(ABA)SAEKI was not
available in radioactive form and Dap(IASA)-YIK(ABA)SAEKI very
inefficiently photoaffinity labeled Kd,
125IASA-YIK(ABA)SAEKI was used for radioactive
photoaffinity labeling experiments.
From 14 independent CTL clones derived from mice immunized
with ANBA-YIK(ABA)SAEKI, seven that exhibited efficient TCR
photoaffinity labeling were selected for further studies. As shown for
a representative experiment in Table I, the
concentration of ANBA-YIK(ABA)SAEKI required for half-maximal lysis of
P815 target cells was in the range of 0.5 (V8 clone) to 150 pM (U3 clone), which is comparable with other
Kd-restricted CTL clones (15, 16). The recognition by all
clones was inhibited by the anti-Kd 1 mAb 20-8-4S,
indicating that they were Kd-restricted (data not
shown).
|
All clones efficiently recognized the derivative lacking the N-terminal
photoreactive group (YIK(ABA)SAEKI), but none detectably recognized the
derivative lacking ABA (YIKSAEKI) except for a very inefficient
recognition by the V17 clone (Table I). Interestingly, all clones
recognized, though some inefficiently, the variant containing acetyl in
place of ABA (YIK(Ac)SAEKI), suggesting that the
N amide bond of K(ABA) may participate
in TCR-ligand binding. Moreover, the derivatives containing alanine or
K(Ac) in place of Lys-259 (YIK(ABA) SAEAI or YIK(ABA)SAEK(Ac)I) were
not detectably recognized by all clones (Table I and data not shown).
Shortening of K(ABA) by one methylene group (YIOrn(ABA)SAEKI) abolished
recognition by all except the U3 and V19 clones, which inefficiently
recognized this derivative. Similarly, shortening of Lys-259
(YIK(ABA)SAEOrnI) abolished the recognition by all but the U3 and V13
clones, which recognized this variant 3- and 100-fold, respectively,
less efficiently (Table I). These results demonstrate that K(ABA) and
Lys-259, but not the N-terminal photoreactive group, were essential
parts of the epitope recognized by these clones and that generally the full spacer length of these side chains was required for efficient antigen recognition.
To obtain information on TCR-Kd contacts, we assessed the
ability of the CTL clones to recognize ANBA-YIK(ABA)SAEKI on L cells expressing mutant Kd molecules. The mutations were single
alanine substitutions of surface exposed residues in the middle parts
of the 1 and
2 helices (e.g. Glu-62, Gln-65, Ser-69,
Gln-72, Gln-149, Glu-154, Tyr-155, and Glu-163). As shown for a
representative experiment in Fig. 1, the recognition of
ANBA-YIK(ABA)SAEKI by all clones, except the V17 clone, was impaired or
abolished by at least one of the Kd
1 mutations E62A,
S69A, or Q72A. In contrast, of the Kd
2 mutations, only
Y155A dramatically affected the recognition by all clones. This effect,
however, is explained in part by reduced peptide derivative binding by
this Kd mutant (24).
TCR Photoaffinity Labeling
For TCR photoaffinity labeling,
covalent Kd-peptide derivative complexes were used, which
were obtained by photoaffinity labeling of soluble Kd with
125IASA-YIK(ABA)SAEKI. This derivative, upon correction for
its reduced Kd binding, was recognized by all clones as
efficiently as ANBA-YIK(ABA)SAEKI (data not shown). Following
incubation of the cloned CTL with Kd-peptide derivative
complexes and photoactivation of the ABA group, the cells were
detergent solubilized and the lysates subjected to immunoprecipitation.
The immunoprecipitates with an anti-TCR mAb migrated on SDS-PAGE under
reducing conditions with an apparent molecular mass of 87-92 kDa (Fig.
2A). Since TCR are composed of
disulfide-linked and
chains, 38-45 kDa each (21), the molecular mass of these materials corresponded to trimolecular complexes consisting of Kd heavy chain (45 kDa), the
peptide derivative (~1,400 Da), and one TCR chain.
To assess the specificity of TCR photoaffinity labeling, cloned W13 CTL were incubated with 125IASA-YIK(ABA)SAEKI in the absence or presence of a 300-fold molar excess of peptide PbCS 252-260 or the Db-restricted peptide Ad5 E1a 234-243 (Fig. 2B, lanes 1-3). Following UV irradiation at 312 ± 40 nm, which activates the IASA and ABA groups, the cells were analyzed as in the previous experiment. The TCR photoaffinity labeling observed in the absence (lane 1) or presence of the Db binding peptide (lane 3), was abolished in the presence of the Kd binding PbCS peptide (lane 2), indicating that TCR photoaffinity labeling required peptide derivative binding to Kd (of CTL) and that free conjugate was unable to detectably label the TCR. The same findings were obtained for the other clones (data not shown).
Analysis of total lysate of W13 cells labeled with
Kd-"125IASA"-YIK(ABA)SAEKI on SDS-PAGE
under reducing conditions showed two labeled species of apparent
molecular masses of 45 and 90 kDa, respectively (lane 4).
These materials correspond to the Kd-peptide derivative
complex and the TCR-ligand complex, respectively. The absence of other
labeled species shows that the TCR photoaffinity labeling was
remarkably selective. The same labeled species were observed following
immunoprecipitation with anti-Kd 3 mAb SF1-1.1.1
(lane 5), which precipitates free as well as ligand
cross-linked with TCR (17). Upon immunoprecipitation with anti-TCR mAb,
only the trimolecular complex was observed (lane 6). This
TCR photoaffinity labeling was inhibited by the anti-Kd
1 mAb 20-8-4S (lane 7), which prevents
Kd-TCR interactions (17), but not by the
anti-Dd mAb 34-4-20S (lane 8). Finally, when
analyzed by SDS-PAGE under nonreducing conditions, labeled material of
approximately 130 kDa was observed (lane 9). This increase
in molecular mass corresponds to the second TCR chain, which is part of
the trimolecular complex under these conditions. These results
demonstrate that the TCR photoaffinity labeling was specific and
required peptide derivative presentation by Kd.
To identify
which TCR chain was photoaffinity labeled, W13 TCR was labeled with
Kd-associated Dap(125ISA)-YIK(ABA)SAEKI. This
conjugate was recognized by all CTL clones as efficiently as
ANBA-YIK(ABA)SAEKI (data not shown) but, due to lacking an N-terminal
photoreactive group, was unable to cross-link to Kd. The
immunoprecipitated TCRs were analyzed by two-dimensional gel
electrophoresis in which the first dimension was IEF and the second
SDS-PAGE. As shown in Fig. 3A, labeled
material with an IP of about 4.9 and an apparent molecular mass of
approximately 43 kDa was observed. Alternatively, W13 cells were
surface radioiodinated and analyzed likewise. The same analysis showed
two labeled materials, one of which migrated very similarly to the one
in the previous experiment while the other had an IP of about 6.5 and
an apparent molecular mass of approximately 44 kDa (Fig.
3B). Since both TCR chains have similar molecular mass
but the IP of chains are acidic (IP 4-5) and those of
chains
nearly neutral (21), these results indicate that the W13 TCR was
photoaffinity labeled selectively at the
chain. The same analysis
was performed with the other clones. As shown in Fig. 3C,
all clones were labeled selectively at the
chain except for the V17
TCR, which was labeled 70% at the
chain and 30% at the
chain.
TCR Sequencing by PCR
PCR using V region-specific primers
showed that four of the seven clones (U4, V8, V19, and W13) expressed
BV1S1A1 encoded chains (Table II, left).
Sequencing indicated that their junctional, CDR3
equivalent regions,
were 11-12 residues long and contained Ser in position 1, Gln in
position 2, Asp or Glu in position 3, Gly in positions 5 and 6, usually
Glu in position 10, and Leu in PC (Table II, left). The
corresponding J
were different, but all, except one (U4), belonged
to the J
2 family. Of the remaining clones, two expressed BV8S1 (U3
and V17) and one expressed BV13S1 (V13). The corresponding CDR3
s
were more diverse but always acidic. The same analysis performed on the
chains indicated that the V
, J
, and CDR3
sequences were
diverse. For the V8 clone, none of the V
-specific primers (22) used
for screening (25) detected an in-frame transcript. Performing the same
analysis on six additional ANBA-YIK(ABA)SAEKI-specific CTL clones
showed a very similar pattern (data not shown). The finding that
specific TCR-
, but not -
, sequences were selected was puzzling
because these TCR were selectively photoaffinity labeled at the
chain (Fig. 3C).
|
To elucidate this paradox, we mapped the photoaffinity
labeled site on a representative TCR, the W13 TCR, which was BV1S1A1 and AV4S10 encoded (Table II). To this end, W13 TCR was photoaffinity labeled with Kd-associated
Dap(125ISA)-YIK(ABA)SAEKI and following reduction and
alkylation, was extensively digested with protease V-8. The resulting
digest fragments were separated by HPLC on a C-18 column. As shown in
Fig. 4A, the major labeled fragments
reproducibly eluted from the column after approximately 33 min. On
SDS-PAGE, this material was homogeneous and migrated with an apparent
molecular mass of about 4,300 Da (Fig. 4F, lane
1). The later eluting labeled materials migrated slower on
SDS-PAGE (apparent molecular masses of about 8,300 Da, 38 min, and
13,000 Da, 44 min, respectively) and were more abundant after shorter
digest periods, suggesting that they were incomplete digest
products.
When trypsin was used for digestion, the major labeled fragments also eluted after about 33 min (Fig. 4B). On SDS-PAGE, this material was homogeneous but migrated slightly faster (apparent molecular mass of about 3,800 Da) than the major labeled V-8 digest fragment (Fig. 4F, lane 2). A second labeled tryptic digest fragment eluted from the HPLC column after 38-39 min and migrated on SDS-PAGE with an apparent molecular mass of about 7,600 Da. This component again was more abundant after shorter digest periods, suggesting that it was an incompletely digested fragment (data not shown).
Digestion of the labeled primary V-8 digest fragment with trypsin
resulted in a new fragment that by HPLC and SDS-PAGE was indistinguishable from the labeled primary tryptic fragment (Fig. 4,
C and F, lane 3). Treatment of the
primary tryptic fragment with V-8 yielded no new product according to
HPLC and SDS-PAGE (data not shown). As shown in Fig. 5,
the W13 TCR V/J
sequence contains only two prolines, Pro-32 and
Pro-41. To assess whether the labeled primary V-8 and tryptic digest
fragments contained prolines, they were treated with endoprotease
Pro-C. As shown in Fig. 4, D and F (lane
5), this enzyme converted the labeled primary V-8 digest fragment
into a new fragment, which eluted from the HPLC column 2 min earlier
and, by SDS-PAGE, had an apparent molecular mass of about 2,000 Da.
Similar results were obtained when the labeled primary tryptic digest
fragment was treated with Pro-C (Fig. 4, E and F,
lane 4).
Since the W13 sequence contains only two prolines (Fig. 5A) and Pro-C is highly specific for proline peptide bonds (28), these results indicate that both labeled primary digest fragments contain prolines. Moreover, the high similarity between both labeled secondary Pro-C digest fragments very strongly suggests that the labeled site was contained in the sequence between Pro-32 and Pro-41 (e.g. segment 33-41). This is consistent with the observed molecular mass and the finding that the same secondary Pro-C digest fragments were observed upon treatment of the minor, later eluting labeled primary V-8 or tryptic fragments (data not shown). If this were correct, the labeled primary V-8 digest fragment would correspond to residues 15-43, and the primary tryptic fragment would correspond to residues 17-39. This is in accordance with the observed apparent molecular mass (Fig. 4F, lanes 1 and 2) but also with the finding that the labeled primary and secondary tryptic digest fragments were very similar, and slightly smaller than the labeled primary V-8 digest fragment. Logically, the photoaffinity labeled site needs to be contained in the sequence common to these fragments, which is the segment 33-39 (Fig. 5A).
Computer Modeling of the W13 TCR and Its LigandTo better
understand in structural terms the data obtained, we built models of
the ligand and the W13 TCR. The TCR model suggests the presence of a
deep hydrophobic pocket between CDR1, CDR2
, CDR3
, and CDR3
(Fig. 6A), which is confined in essence by
the side chains of CDR3
Ser-99 and Ala-100 and CDR3
Ser-94 and
Leu-102 and the V
residues Asn-33 and Phe-35 (C-strand) and Ala-92
(F-strand) (Fig. 6, B and C). As suggested by
docking experiments, this pocket, upon small structural changes, can
avidly bind the K(ABA) side chain of the ligand (Fig. 6, A
and C, and data not shown). The main forces driving this
interaction include: i) hydrophobic interactions (e.g. the
pocket and K(ABA) side chain evading contact with water), ii)
significant Van der Waals interactions due to high complementarity of
pocket and side chain, iii) 8
-8
interaction of the ABA azido substituent with the phenyl moiety of V
Phe-35, and iiii) hydrogen bond formation of the ABA carbonyl oxygen, the most polar atom of this
side chain, with CDR3
Ser-94 (Fig. 6B). This prediction is consistent with the finding that the photoaffinity labeled sites
were contained in the V
segment 33-39 (Figs. 4 and 5).
According to our ligand model, the K(ABA) side chain of
Kd-bound ANBA-YIK(ABA)AEKI is very extended and "leans
over" to the Kd 2 helix (Fig. 6D). A very
similar orientation was obtained when the ligand was modeled alone or
together with the W13 TCR (data not shown). The Kd residues
Glu-62, Ser-69, and Tyr-155, which upon alanine substitution dramatically affected the antigen recognition by the W13 clone (Fig.
1), are located on the surface of the
1 and the
2 helices, respectively.
While the available crystallographical coordinates permitted modeling
of the TCR framework, including the hydrophobic pocket, with good
accuracy, conclusive modeling of CDR3 loops was elusive because of the
high number of possible, energetically equivalent conformers. We
therefore modeled these loops by using the ligand surface as
"template" to define loop conformations that provide the lowest
energy of the TCR-ligand complex. These docking experiments were based
on the assumptions that 1) the ligand K(ABA) side chain inserts in the hydrophobic pocket of the W13 TCR, 2) the Kd
residues Glu-62, Ser-69, and Tyr-155, but not Gln-65, Gln-72, Gln-149,
Glu-154, or Glu-163, interact with the TCR, and 3) Asp-95 or Glu-101 of
CDR3 interact with PbCS Lys-259. The latter assumption was made
based on the finding that the W13 TCR (and most other TCR of that
specificity) has two acidic residues in CDR3
, but none in CDR1 and
CDR2 of both chains (Fig. 5). These experiments suggest that the W13
TCR interacts with its ligand in the orientation shown in Fig.
6A, in which the C-terminal portion of the
Kd-bound peptide is located underneath CDR3
, and the
N-terminal portion is underneath CDR3
. It is noteworthy that the
helices of Kd, especially the
2 helix, are slanted, and
the W13 TCR has a complementary surface contour, which provides a
maximal surface contact in this orientation (data not shown).
To study TCR-ligand interactions by TCR photoaffinity labeling, we derived CTL clones from mice immunized with the PbCS peptide derivative ANBA-YIK(ABA)SAEKI. As in previous studies with other photoreactive PbCS peptide derivatives (15, 16), ANBA-YIK(ABA)SAEKI-specific CTL were readily obtained. The "photoprobe"-specific CTL described here, as well as those described previously, displayed all the hallmarks of antigen recognition by "conventional" CTL, including MHC restriction and dependence on auxiliary molecules (i.e. CD8 and LFA-1) (Fig. 2 and data not shown) (17, 29). Similarly, numerous reports described CD8+ as well as CD4+ T cells that recognize antigens modified with haptens such as trinitrophenyl, benzoarsonate, or fluorescein but also with carbohydrates (13, 30-32). The epitopes recognized by these T cells, as far as is known, are MHC bound peptides that contain nonpeptidic entities. Thus, while antigen recognition by T cells is MHC restricted, it is not limited to conventional peptides but clearly can include a vast array of structures. In several cases, such T cell reactivities have been shown to play a role in disorders such as drug allergies or delayed type hypersensitivities (33).
The epitope recognized by ANBA-YIK(ABA)SAEKI-specific CTL clones included two peptide derivative residues, K(ABA) and PbCS Lys-259 (Table I). The latter residue was also essential for the recognition of the PbCS 252-260 peptide by most PbCS-specific CTL clones (16). Similarly, the antigen recognition by IASA-YIPSAEK(ABA)I-specific CTL clones required K(ABA) in position 8 (15). These findings demonstrate that for the PbCS system, the residue in position 8 is a primary TCR contact residue. This is consistent with computer modeling suggesting that this ligand residue is fully solvent exposed (Fig. 6D and Ref. 15). However, the three systems differed regarding the residue in position 4. For PbCS and IASA-YIPSAEK(ABA)I-specific CTL PbCS, Pro-255 was a secondary TCR contact residue (15, 16), but for ANBA-YIK(ABA)SAEKI-specific ones, K(ABA), in this position, was essential (Table I). Since the N-terminal photoreactive group was not required for antigen recognition by ANBA-YIK(ABA)SAEKI and IASA-YIPSAEK(ABA)I-specific clones (Table I and Ref. 15), the epitopes recognized by these families of clones essentially differed by the absence or presence of K(ABA) in positions 4 and 8, respectively.
The TCR expressed by the three families of clones were also different.
Those expressed by PbCS-specific CTL clones were highly diverse both in
terms of CDR3 sequences and TCR gene element usage, except that 57% of
the clones expressed BV3S1A1 (25). In contrast, about 80% of
IASA-YIPSAEK(ABA)I-specific clones expressed BV1S1 encoded chains that were paired with J
TA28 encoded
chains (15). On the
other hand, the ANBA-YIK(ABA)SAEKI-specific CTL also preferentially
expressed BV1S1, though less frequently, but the
chains lacked any
apparent sequence consensus (Table II).2
Remarkably, however, the ANBA-YIK(ABA)SAEKI-specific TCR, unlike the
IASA-YIPSAEK(ABA)I-specific ones, were selectively labeled at the
chain (Fig. 3). Peptide mapping showed that the W13 TCR photoaffinity
labeling took place in a site-specific manner (Fig. 4), which implies
that the ABA group specifically bound to this TCR.
As suggested by computer modeling, the W13 TCR ligand binding site
contains a deep hydrophobic pocket, which is located and structured
such that it can avidly bind the ligand K(ABA) side chain (Fig. 6).
This is consistent with the localization of the photoaffinity labeled
site in the V C-strand segment 33-39, which contains Phe-35, that
according to our docking experiments intimately interacts with the ABA
azide substituent (Figs. 4 and 6, B and C). In
accordance with that is the finding that the derivative lacking this
substituent (YIK(BA)SAEKI) was recognized by W13 CTL less
efficiently.2 In addition, the most polar atom of this side
chain, the carbonyl oxygen of the ABA group, according to our modeling
forms a stabilizing hydrogen bond with CDR3
Ser-94, which is in
agreement with the observation that W13 CTL recognized, though
inefficiently, the variant YIK(Ac)SAEKI (Table I).
The binding of the K(ABA) side chain in a hydrophobic pocket,
formed mainly by CDR3 and V
framework residues, which are more
conserved than CDR residues (34, 35), may explain why these TCR were
preferentially labeled at the
chain even though they lacked any
apparent sequence consensus (Table II). The finding that a side chain
of an MHC-bound peptide interacts with TCR V
framework residues is
not in agreement with the current concept of TCR-ligand interactions,
which postulates that such residues interact primarily with CDR3 loops
(6). It is, however, important to note that the K(ABA) side chain is
considerably longer than a conventional amino acid side chain (Fig.
6D). This long spacer is required for the ABA group to
interact with the TCR, as demonstrated by the observation that
shortening of this side chain by only one methylene group (1.5 Å)
abolished the antigen recognition by the W13 as well as most other CTL
clones (Table I). Moreover, the K(ABA) side chain is remarkably
hydrophobic and hence preferentially interacts with nonpolar domains on
TCR. Since these TCR were selected for avid binding of the
photoreactive ligand, it can be expected that their ligand binding
includes an avid binding of the K(ABA) side chain. The accommodation of
the ABA group in a hydrophobic pocket, as proposed by our modeling,
constitutes such a binding principle (Fig. 6,
A-C).
These findings are reminiscent of a previous study, in which we showed
that a BV1SA1/JTA28 encoded TCR, specific for IASA-YIPSAEK(ABA)I, utilized a similar principle to bind the K(ABA) side chain (15). However, in this case, the hydrophobic pocket was located and formed by
the BV1S1A1 C- and C
-strands and the J
TA28 encoded portion of
CDR3
and adjacent G-strand residues (15). Based on these findings,
we propose that TCR can express hydrophobic domains in the indicated
locations; since they are formed by TCR framework and CDR3
residues,
their shape and physicochemical nature may vary significantly according
to TCR sequences. While normal amino acid side chains of MHC-bound
peptides are unlikely to interact with these domains, we suggest that
they enable TCR to efficiently and specifically bind epitopes
containing modified side chains. Such side chains usually are long and
often amphiphatic or hydrophobic, which allows them to efficiently
interact with these sites.
It remains to be explained why ANBA-YIK(ABA)SAEKI-specific TCRs were
selectively photoaffinity labeled at the chain, while the
IASA-YIPSAEK(ABA)-specific ones were labeled at the
chain or both
chains (Fig. 3) (15). This labeling pattern may be explained by either
of two different orientations of TCR-ligand interactions. i) The
Kd bound peptide runs approximately diagonal across the TCR
binding site, such that K(ABA) in position 8 interacts mainly with the
and in position 4 with the
chain (Fig. 6A). Strong
evidence for this orientation has been reported by Sant'Angelo
et al. (10) and Sun et al. (37). ii)
Alternatively, the MHC-bound peptide could be located underneath
CDR3
and CDR3
, e.g. rotated by 30-40° relative to the
orientation shown in Fig. 6A, in which case the orientation
of the K(ABA) side chain determines with which TCR chain the ABA group
preferentially interacts. This orientation corresponds to the one
predicted on theoretical grounds (6) and by studies in which TCR single
chain transgenic mice were immunized with peptide variants (9).
The system described here and the one described previously (15) allows assessment of TCR-ligand interactions by TCR photoaffinity labeling. This permits rapid and conclusive mutational analysis of TCR-ligand interactions but also makes possible the preparation of covalent TCR-ligand complexes for x-ray crystallographic studies. In addition, as TCR photoaffinity labeling is applicable on living cells, these systems are very suitable for structure-function studies and assessment of co-receptor participation in TCR-ligand interactions (29).
We thank Drs. Graham Bentley and Roy Mariuzza for providing the coordinates of TCR structures, Christian Jaulin and Philip Kourilsky for Kd-transfected L cells, Jean Gagnon for endoprotease Pro-C, and Anna Zoppi for preparing the manuscript.