By
§
§
¶
¶
From the * Howard Hughes Medical Institute, Department of Medicine, National Jewish Medical and
Research Center, and § Department of Immunology,
Department of Biochemistry, Biophysics and
Genetics, and ¶ Department of Medicine, University of Colorado Health Sciences Center, Denver,
Colorado 80206
The /
T cell receptor (TCR) recognizes peptide fragments bound in the groove of major
histocompatibility complex (MHC) molecules. We modified the TCR
chain from a mouse
T cell hybridoma and tested its ability to reconstitute TCR expression and function in an
chain-deficient variant of the hybridoma. The modified
chain differed from wild type only
in its leader peptide and mature NH2-terminal amino acid. Reconstituted cell surface TCR complexes reacted normally with anti-TCR and anti-CD3 antibodies. Although cross-linking
of this TCR with an antibody to the TCR idiotype elicited vigorous T cell hybridoma activation, stimulation with its natural MHC + peptide ligand did not. We demonstrated that this
phenotype could be reproduced simply by substituting the glutamic acid (E) at the mature NH2
terminus of the wild type TCR
chain with aspartic acid (D). The substitution also dramatically reduced the affinity of soluble
/
-TCR heterodimers for soluble MHC + peptide molecules in a cell-free system, suggesting that it did not exert its effect simply by disrupting TCR
interactions with accessory molecules on the hybridoma. These results demonstrate for the first
time that amino acids which are not in the canonical TCR complementarity determining regions can be critical in determining how the TCR engages MHC + peptide.
The structural basis of antigen recognition by antibody
molecules has been intensely studied using x-ray crystallography. Crystal structures of a large array of antibodies
and antibody-antigen complexes have demonstrated that the
hypervariable regions of Ig genes encode solvent-exposed
loops that cluster to form the antigen-binding site of the Ig
V domains. These loops are known as CDRs. Analysis of
Ig crystal structures and CDR sequences has shown that the
spatial orientation and conformation of the loops is influenced by two factors.
The first concerns the CDR itself. Each of the CDR
loops appears to adopt a limited range of main chain conformations despite their diverse amino acid sequences (1).
These conformations are shaped by a small number of relatively well-conserved amino acid residues within the loops.
The second factor is the V domain scaffolding upon which
the CDRs rest. It consists of two The In this paper, we describe a mutation in the NH2-terminal V Cell Lines.
The mouse T cell hybridoma used in these studies
was 2B10.D2O-22.3 (22.3) specific for chicken OVA peptides
323-339 or 327-339 presented by IAd (IAd/OVA). It was generated by fusing the AKR thymoma fusion partner BW-1100.
129.237 (BW Two-color Flow Cytometry.
T cell hybridomas were stained for
cell surface molecules and analyzed by flow cytometry as previously described (21) using the following mAbs: KJ1-26 (22) is
specific for the idiotype of the TCR on DO-11.10 and 22.3, and
H57-597 (23), 145-2C11 (24), and GK1.5 (25) are specific for
mouse TCR C T Cell Hybridoma Stimulation Assays.
T cell hybridoma activation in response to TCR ligation was assayed by measuring IL-2
secretion, as described previously (26). Stimulation assays were
performed in 96-well flat-bottomed microtiter plates. In peptide
stimulations 105 IAd-expressing A20-1.11/B5 B cell lymphoma cells
(27) were added per well with different concentrations of OVA
327-339. For anti-TCR mAb stimulation, Immulon-3 (Dynatech Labs. Inc., Chantilly, VA) microtiter plates were coated
overnight at 4°C with serial dilutions of protein A-purified KJ126 in PBS. Peptide and antibody stimulations both used 105 hybridoma T cells/well and were incubated for 16-30 h at 37°C. IL-2 production was quantitated using the IL-2-dependent cell line HT-2 (28) and measurement of HT-2 survival by the oxidation of (dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(29).
Characterization of the V
DO
Production of Soluble TCRs and IAd Protein Covalently Bound to
Chicken OVA 327-339.
XhoI/AccIII fragments of DO BIAcore Measurements of TCR Binding to IAd-OVA.
The BIAcore system was used to assess soluble IAd-OVA binding to
TCRs. The high affinity anti-C To study the effects of mutations in the TCR The infectants were screened for cell surface TCR expression by staining with mAbs to the DO TCR idiotype
and CD3
To determine whether the structural integrity of the
TCR complexes on the infectants was also reflected in
functional competence, the infectants were cultured in
plastic wells coated with antiidiotype mAb. In response to
this TCR ligation and cross-linking stimulus, the infectants
secreted IL-2 in a manner similar to that of the wild-type
hybridoma, 22.3 (Fig. 3). This suggested that the TCR complexes containing the DO Since DO
To
identify individually the contributions of leader peptide and
NH2 terminus to DO
In contrast, the infectants were readily divided into two
groups based on their ability to respond to IAd/OVA (Fig.
6). Regardless of the leader peptide they used, infectants expressing D at the NH2 terminus of their TCR
These experiments demonstrated that the leader peptide sequence
did not affect the function of the DO To test this hypothesis, genes encoding soluble DO The simplest explanation for our results was that the
substitution of D for E at the NH2 terminus of DO Anti-C
We have demonstrated that an E to D change in the
NH2-terminal amino acid of a mouse TCR The E to D substitution could have affected MHC + peptide responsiveness either directly, by disrupting a contact between this amino acid and the ligand, or indirectly,
by altering the shape or position of amino acids on one of
the receptor CDR loops. The recent crystal structures of
TCR V
In all three structures, the V Support for this notion comes from the study of Ig structures. One study comparing Ig crystal structures concluded
that the conformation and position of CDR2 of the Ig
heavy chain is largely shaped by the nature of the side chain
on framework residue 71 (2). In another study, a spontaneous variant of an antidigoxin antibody with 580-fold less
affinity for digoxin than wild type was found to have a substitution from S to R at position 94 in its heavy chain, a
residue predicted to lie in the framework at the base of
CDR3 (48, 51). Computer modeling suggested that the
substitution increased hydrogen bonding between CDR3
of the heavy chain and CDR2 of the light chain so that the
digoxin binding surface of the Ig was altered. Interestingly,
most of the substitution's effect was reversed if two residues
from the NH2 terminus of the heavy chain were removed.
Modeling suggested that the deletion increased the solvent
accessibility of R94, destabilizing the aberrant hydrogen
bonding and returning the CDR loop structures to wild
type.
In the two TCR + MHC class I crystal structures, the
side chain of the V Although further work will be necessary to confirm the
generality of our results with this particular sheets packed face to
face and anchored by a structurally conserved core of "framework" residues. The conformations of CDR loops are partially determined by the manner in which they pack with
the side chains of certain framework residues (1, 2). An understanding of these factors has resulted in a limited ability
to predict CDR loop conformations of Igs of unknown
crystal structure from the sequences of their heavy and light
chain genes (1). This is a first step towards the engineering
of antibodies specific for antigens of interest.
/
-TCR recognizes peptide fragments bound in
the groove of MHC molecules. The structural basis of this
recognition is understood in far less detail than that for Igs,
largely because soluble versions of these naturally membrane-bound structures have only recently been developed.
Crystal structures of MHC class I (3) and class II (8)
molecules with single peptides bound in their grooves have
provided a detailed picture of the ligand with which TCRs
interact. Crystal structures of one mouse TCR
chain (11),
one mouse TCR V
domain (12), and two
/
-TCRs bound to their class I MHC + peptide ligands (13, 14),
verified predictions (15) that the ligand binding site of
the TCR consists of Ig-like CDR loops supported by a
dual
sheet framework, but has not yet provided enough
information to allow the construction of detailed models of
the CDR loop architecture or the examination of the role
of TCR framework residues in shaping it.
amino acid of a mouse
/
-TCR that alters its
ability to bind to its MHC + peptide ligand. We discuss
how this non-CDR residue may play an important role in
TCR specificity.
/
; 18) to lymph node cells from a B10.D2 mouse
immunized with OVA 327-339 (Kushnir, E., unpublished data).
The TCR
and
chains of 22.3 are identical in amino acid sequence to those of DO-11.10, a previously described T cell hybridoma with the same specificity (19, 20). Therefore, these chains
are designated DO
and DO
in this paper. Unlike DO-11.10,
however, 22.3 did not express any other functional TCR-
or -
genes. 22.3 was recloned to isolate two subclones, 22.3.111 and
22.3.145, which bear lower levels of surface TCR. In addition, a
spontaneous TCR
chain loss variant of 22.3 was isolated,
22.3
. This variant expresses DO
mRNA, but does not contain DNA encoding DO
and therefore was useful as a recipient
for ( chain transfection studies.
, CD3
, and CD4, respectively. In brief, 2-5 × 105 hybridoma cells were incubated with biotinylated KJ1-26 or
H57-597 at 4°C for 20 min. Cells were washed three times and
incubated at 4°C for 20 min with FITC-conjugated 145-2C11 or
GK1.5 and streptavidin R-conjugated phycoerythrin (Sigma Chemical Co., St. Louis, MO). Finally, cells were washed three times
and two-color fluorescence data was acquired on either an EPICS
C or a Profile flow cytometer (Coulter Corp., Hialeah, FL).
13.1 leader from 22.3.
Using an anchored PCR strategy modified from Loh et al. (30), DNA encoding the V
13.1 (AV13S1) leader was cloned to determine the unknown sequence of its 5
end. Total RNA was prepared from the
22.3 hybridoma using the Ultraspec reagent (Biotecx Labs., Houston, TX). Oligo-dT-primed first strand cDNA was synthesized using the SuperScript reverse transcriptase cDNA synthesis kit (GIBCO BRL, Gaithersburg, MD) and was tailed at the 5
end
with dG residues using terminal deoxynucleotidyl transferase (International Biotechnologies Inc., New Haven, CT). TCR
chain gene sequences were PCR amplified from tailed cDNA
using the anchored sense primer 5
-ATCGAGTCGACGCCCCCCCC-3
and the antisense primer 5
-CCCCAGACAGCCGTCTTGACG-3
, synthesized at the National Jewish Molecular Resource Center (Denver, CO). The latter primer anneals to
an insertion in the 3
untranslated region of TCR
chain transcripts that is present in C57 mouse strains, but not AKR or
BALB/c strains (31, 32, and Seibel, J., unpublished data). This
avoided amplification of the nonfunctional TCR
chain transcripts contributed to the 22.3 hybridoma by the AKR thymoma
fusion partner BW
/
(33, 34). This anchored PCR product
was reamplified using the same anchored sense primer and the antisense primer 5
-TCTCGAATTCAGGCAGAGGGTGCTGTCC-3
(35), yielding a product that was digested with SalI and
EcoRI and cloned into the pTZ18R vector (Pharmacia, Piscataway, NJ) for sequencing. The complete cDNA sequence of the
V
13.1 leader and its 5
untranslated region are presented in Fig. 1.
Fig. 1.
Complete nucleotide sequence of the V13.1 leader and its
5
untranslated region. The sequence of the V
13.1 leader was assembled from six independently cloned PCR products of different lengths, which
were obtained by anchored reverse transcriptase PCR from the B10.D2derived T cell hybridoma, 22.3. Deduced amino acid residues are shown.
Underlined triplets in the 5
untranslated region indicate stop codons,
which are found in all three reading frames. The V
11.2 leader is shown
for comparison. Assignment of the boundary between the leaders and the
mature variable domains is based on standard mouse TCR
chain sequence alignments (54).
[View Larger Version of this Image (27K GIF file)]
Chain Gene Constructs and Expression Vectors.
We used
PCR techniques (36, 37) to construct four versions of the V
portion of DO
chain gene using either the V
11.2 (AV11S2) or
V
13.1 (AV13S1) leader peptide and having a predicted NH2terminal amino acid of either aspartic acid (D) or glutamic acid (E).
These are designated DO
L11terD, DO
L11terE, DO
L13terD, and DO(L13terE (identical to wild-type DO
(Fig. 2). These
were cloned in pTZ18R fused in frame to the mouse C
gene
using a XhoI site at the 5
-end of the V
segment and an introduced AccIII site at the 5
-end of C
(36), such that the final
complete
chain was flanked by EcoRI sites. To express these
variant DO
constructs in the 22.3
hybridoma, the EcoRI
fragments were isolated and cloned into the EcoRI site of the murine retrovirus-based vector, LXSN (38). Using methods modified from Miller et al. (38), DO
RNAs were packaged into retroviruses in the GP&env AM12 (39) and PA-317 (40) cell lines,
and were introduced into the 22.3
hybridoma by infection.
Infectants with stably integrated DO
genes were selected and
maintained with culture medium containing 1-1.2 mg/ml G418
(Geneticin; GIBCO BRL).
Fig. 2.
DO chain variant constructs. Circled letters indicate the predicted NH2-terminal amino acid of the mature
chain.
[View Larger Version of this Image (37K GIF file)]
L11terD
and DO
L13terE were cloned in frame with a truncated gene for
mouse C
in a previously described baculovirus transfer vector
that also encodes the soluble DO-11.10(
1,3) TCR
chain (36).
Recombinant baculoviruses prepared from these vectors coexpressing the TCR-
and -
genes were used to infect Hi 5 insect cells (Invitrogen, San Diego, CA). Soluble heterodimers
were purified from cell supernatants by immunoaffinity chromatography on an antiidiotype column, followed by a HiLoad 26.60 Superdex200 size exclusion column (Pharmacia). The proteins were further purified to a single detectable species using Mono Q
ion exchange chromatography (Pharmacia). IAd-OVA, a soluble
ligand for the DO
/
-TCR that consists of a soluble IAd molecule genetically attached to the OVA peptide via a flexible peptide linker to the IAd
chain NH2 terminus, was produced in
baculovirus as previously described (41). It was purified by immunoaffinity chromatography on an anti-IAd mAb (M5/114, reference 42) column, and further purified on a HiLoad 26.60 Superdex 200 column. Gels revealed that these procedures yielded
preparations of monomeric heterodimers of both the TCR and
class II proteins with no detectable contamination of either protein with aggregates or homodimers.
mAb, H57-597, was immobilized in flow cells of a BIAcore biosensor CM-5 chip using standard amine coupling chemistry. Flow cells were injected with
solutions of various soluble TCRs at 5 µg/ml until binding to the
anti-C
antibody was saturated. This captured TCR increased
surface plasmon resonance signals by ~2,000 resonance units.
Since the captured TCR dissociated very slowly, we could detect
its binding to subsequently injected IAd-OVA. Solutions of IAdOVA (2.5, 5, or 10 µM in phosphate-buffered saline containing 5 mM azide and 0.005% P20 surfactant) were injected for 1 min at
a flow rate of 20 µl/min. The TCR-bound IAd-OVA was then
allowed to dissociate for several minutes. Between injections, the
anti-C
mAb was resaturated with TCR. To control for signal
due to the bulk refractive index of the IAd-OVA in solution, or
for any differences in the refractive index of the buffers used, an
identical set of injections were performed in a flow cell containing captured free DO
chain, rather than
/
-TCR. On and off
rates for binding of IAd-OVA were calculated using the BIAcore
software.
Expression of a TCR Chain in an
Chain-deficient
Mouse T Cell Hybridoma.
chain on MHC + peptide recognition
by T cells, we used a mouse T cell hybridoma, 22.3, which
could be stimulated by either IAd class II MHC molecules
bound to chicken OVA 327-339 (IAd/OVA) or by the
anti-TCR idiotype mAb, KJ1-26 (22). In an initial experiment, receptor expression in an
loss variant of this hybridoma, 22.3
, was restored by infection with a retrovirus
carrying a version of the full-length V
13.1 bearing DO
chain gene. This construction, DO
L11terD, differed from
the natural DO
chain in two respects. First, it contained a
V
11.2 leader (L11) in lieu of the then unknown native
V
13.1 leader (L13). Second, after cleavage of its leader
peptide, the mature NH2 terminus of the DO
L11terD chain was aspartic acid (terD), instead of the glutamic acid
(terE) of wild-type DO
. This conservative substitution was
introduced to maintain a convenient cloning site. It was
not expected to affect TCR specificity, since we had previously shown that it had no detectable effect on the binding
of a number of anti-TCR mAbs, including KJ1-26, an mAb
specific for the receptor idiotype (Kappler, J., unpublished
data).
chain. Their levels of idiotype-reactive TCR
were slightly lower than that of the parental hybridoma,
22.3, but were equal to or greater than those on two low
TCR-expressing subclones of 22.3, 22.3.111, and 22.3.145 (Fig. 3). Cell surface levels of CD3
suggested proper TCR
association with CD3 components (data not shown). These
staining results suggested that the DO
L11terD protein
folded properly, associated normally with the TCR
chain
and CD3 components, and reconstituted cell surface TCR
complexes of normal structure.
Fig. 3.
Infectants bearing the variant TCR chain DO
L11terD
respond to immobilized antiidiotype mAb as well as the parental hybridoma, 22.3, does. 22.3.111 and 22.3.145 are low TCR expressing subclones of 22.3. Data are representative of multiple experiments. TCR level indicates the mean linear fluorescence of antiidiotype mAb staining
of the hybridomas.
[View Larger Version of this Image (19K GIF file)]
L11terD chain were sufficient for normal ligation-induced signaling within the T cell
hybridoma.
Chain with the DO
L11TerD
Chain Resulted in a Hybridoma with Dramatically Reduced
Ability to Respond to IAd/OVA.
L11terD-containing TCR heterodimers mediated a vigorous IL-2 response to anti-TCR antibody stimulation, it was surprising that they did not reconstitute good IAd/OVA reactivity. As
shown in Fig. 4, DO
L11terD infectants responded much
more poorly to IAd/OVA than did the parental hybridoma,
22.3. Since the TCR
chain in these infectants differed
from that of 22.3 only in its leader peptide and the substitution of D for E at the mature NH2 terminus, one or both of
these changes must have caused the impaired responsiveness. The amino acid sequence of the leader might, for example, have altered the site at which the leader is trimmed
from the mature protein. Such an effect has been described
in several proteins (43), including an antidigoxin antibody in which single substitutions in the heavy chain leader
peptide changed the length of the mature chain by up to 10 residues (48). Some of these length changes were sufficient
to change the affinity of the antibody. An alternative hypothesis was that the NH2 terminus of the TCR chain influenced directly or indirectly TCR interaction with MHC + peptide.
Fig. 4.
Infectants bearing the variant TCR chain DO
L11terD
respond poorly to IAd/OVA stimulation. cOVA 327-339 peptide was
presented to the hybridomas by the IAd-expressing B cell lymphoma,
A20. Data points indicate the means of duplicate wells in one representative experiment.
[View Larger Version of this Image (16K GIF file)]
Chains with E at the Predicted NH2 Terminus,
Regardless of V
11.2 or V
13.1 Leader Peptide Usage.
chain function, we generated four
types of 22.3
infectants with each possible combination
of L11, L13, terD, and terE (Fig. 2). The previously uncharacterized L13 leader was cloned from 22.3 (Fig. 1, Materials and Methods). Infectant clones that expressed comparable levels of cell surface TCR were selected by staining
with antiidiotype mAb. Four clones of each type were tested for their ability to respond to antiidiotype antibody
and to IAd/OVA. All four types of infectants possessed
comparable signal transduction capacity, since they produced similar levels of IL-2 when stimulated with immobilized antiidiotype antibody (Fig. 5). We do not know why
one DO(L13terD infectant responded more poorly than
the other three.
Fig. 5.
Infectants bearing
any one of four DO TCR chain variants respond equally
well to immobilized antiidiotype mAb. Data points represent
the means of two independent
experiments with duplicate wells.
Error bars indicate standard errors
of the mean. Four infectants of
each variant type were tested.
TCR levels of all infectants varied by less than twofold.
[View Larger Version of this Image (29K GIF file)]
chains
(DO
L11terD and DO
L13terD) responded very poorly.
By contrast, infectants expressing NH2-terminal E (DO
L11terE and DO
L13terE) responded strongly.
Fig. 6.
Infectants bearing
variant DO chains with D, but
not E, at their predicted NH2
termini respond poorly to IAd/
OVA. Data points represent the
means of three independent experiments with duplicate wells,
except for those at 250 and 500 µg/ml OVA which represent
only two such experiments. Error bars indicate standard errors
of the mean. Four infectants of
each type were tested. TCR levels of all infectants varied by less
than twofold.
[View Larger Version of this Image (23K GIF file)]
Chains Produced
as Soluble Proteins in Baculovirus Were as Predicted.
chain variants, and highlighted the importance of the predicted NH2 terminus
of the mature protein for IAd/OVA responses. However, it
was still formally possible that the E and D containing
chains might have had their signal peptides cleaved at different sites. This would have made the two types of chains
different in length. Some structural aspect of the length disparity could have directly affected the interaction of the TCR with MHC + peptide, but not with anti-TCR antibodies.
chains
with predicted NH2 termini of either E or D were coexpressed in insect cells with a gene encoding a soluble DO
chain. Purified DO
/
heterodimers were sequenced by
Edman degradation. Both DO
variants were found to
bear the predicted NH2-terminal amino acid, E for DO
terE and D for DO
terD. Since insect signal peptidases typically cleave at the same site as their mammalian counterparts (49), we concluded that TCR
chains in our DO
terE and DO
terD infectant hybridomas were identical in
length, differing only in the identity of the amino acid at
their NH2 termini. This difference alone must have been
responsible for their contrasting responses to IAd/OVA.
Chain Affects the Affinity of the DO TCR for MHC + peptide.
directly altered the ability of the TCR to bind to IAd/OVA.
However, it was also possible that the substitution acted indirectly. For example, it could have disrupted TCR interactions with a T cell accessory molecule such as CD4. Such
an interaction may not have been required for activation of
the hybridoma during stimulation with antiidiotype antibody, where large numbers of TCRs were ligated. However, it may have been critical for responses to IAd/OVA
stimulation, where only a few TCRs on each hybridoma
were engaged. To distinguish between these mechanisms,
we used surface plasmon resonance to measure directly the
binding of soluble IAd-OVA (40) to DO
/
heterodimers.
mAb was used to immobilize DO
/
heterodimers containing either DO
terE or DO
terD chains in
separate flow cells of a biosensor chip. A flow cell in which
the free DO
chain was immobilized was used as a negative control. Various concentrations of IAd-OVA were
passed through the flow cells and the binding kinetics followed (Fig. 7). Obvious binding of IAd-OVA to a TCR
containing DO
terE was seen at all class II concentrations. The interaction had a relatively slow on rate, ka = 1.60 × 103 M
1s
1, and a fast off rate, kd = 0.05s
1, resulting in
a dissociation constant (KD) of 31 µM. These kinetic and
thermodynamic constants are similar to those observed for other TCRs binding to MHC class II + antigen complexes
(50). In contrast, IAd-OVA bound very poorly to a TCR
containing DO
terD. Very weak specific binding detected
only at the highest concentration of IAd-OVA. This suggested a dissociation constant >300 µM. The results showed
that the NH2 terminal E to D substitution in DO
disrupted TCR binding to class II + peptide in the absence
of accessory molecules. We concluded that N terminal E
must play a direct role in the interaction of this TCR with
IAd/OVA.
Fig. 7.
Substitution of D for E at the DO chain NH2 terminus
lowers the affinity of cell-free
/
-TCR for IAd-OVA. Various concentrations of IAd-OVA were injected in flow cells with immobilized DO
/
TCRs bearing DO
chains with either D or E at the NH2 terminus as
described in the Materials and Methods. A flow cell with immobilized
free
chain from this receptor was used as a control for signal from protein in solution and buffer differences.
[View Larger Version of this Image (20K GIF file)]
chain significantly altered the affinity of the
/
-TCR heterodimer
for its MHC + peptide ligand. This conservative change
simply shortened the amino acid side chain by a single methylene group without changing its negative charge. This
effect did not result in a gross alteration in TCR structure,
since detection of the TCR complexes by anti-TCR mAbs was unaffected by the substitution. Moreover, overall TCRmediated signal transduction was not impaired by the substitution, since hybridomas bearing it responded normally
to TCR cross-linking by immobilized antiidiotypic mAb.
s both free and as part of TCR + MHC complexes suggest that either of these possibilities is feasible
(Fig. 8, references 12). These structures include a free
V
domain, mouse V
4(AV4S1), and two TCRs bound
to their MHC class I/peptide ligands. These latter TCRs
contained mouse V
3 (AV3S1) and human V
2(AV2S1).
Fig. 8.
Position of the V NH2 terminus in three TCR crystal
structures. The program Molscript was used to create a ribbon representation of three V
elements based on their crystal structures. In each case, a
wire frame representation of the NH2-terminal amino acid is shown. The
mouse V
4 structure (12) was of a V
dimer in the absence of V
. The
mouse V
3 (13) and human V
2 (14) structures were of complete
/
TCRs. The views are of the V
s with their solvent exposed faces toward
and their V
interaction surfaces facing away from the reader.
[View Larger Version of this Image (43K GIF file)]
NH2-terminal amino acid
is solvent exposed and in contact with the V
strands
preceding V
CDR1 and following V
CDR3. It is easily
conceivable that a mutation in this amino acid could alter
CDR1 or CDR3 conformation. This is particularly evident in the mouse V
4 structure (12), where the side chain
carbonyl group of the NH2-terminal aspartic acid interacts
intimately with both
strands.
NH2-terminal amino acid does not
appear to make direct contacts with the MHC ligand and,
in fact, the side chain of the NH2-terminal lysine of human
V
2 appears disordered in the structure (14). However, the
free NH2-terminal amino group of the V
2 chain itself is
connected by a salt bridge to a conserved glutamic acid in
the
helical region of the MHC class I
1 domain (14).
Such a salt bridge is not found in the TCR + MHC class I
structure containing mouse V
3 (13), and probably cannot
be a general feature of TCR + MHC class II complexes
since class II lacks the conserved glutamic acid residue in
the
helix of its
1 domain. However, the overall positions of the V
NH2-termini in the crystals suggest that depending on the exact orientation and pitch of the TCR on
its MHC ligand, direct interaction between the V
NH2terminal amino acid side chain and the MHC ligand may not be an infrequent feature of the complex.
/
-TCR, our
findings suggest that gene constructs used to express soluble
TCRs for use in binding assays and x-ray crystallography
must be designed carefully. Substitutions at non-CDR residues must be introduced with the knowledge that they
have the potential to disrupt MHC + peptide recognition.
A detailed understanding of the role of TCR framework
residues in MHC + peptide recognition will be difficult to
attain until numerous TCR and TCR + MHC + peptide
crystal structures have been solved.
Address correspondence to Dr. Philippa Marrack, National Jewish Center for Immunology and Respiratory Medicine, Goodman Bldg., 5th Floor, 1400 Jackson St., Denver, CO 80206.
Received for publication 10 February 1997 and in revised form 31 March 1997.
We thank Dr. Serge Candeias for assistance with the anchored PCR approach used to clone the V13.1
leader from 22.3. We thank Dr. Daved Fremont for helping us to interpret TCR crystal structures. We are
grateful to Dr. Anthony Vella for advice and encouragement.
This work was supported by United States Public Service grants AI-17134, AI-18785, and AI-22295.
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