By
From the * Center for Advanced Research in Biotechnology, University of Maryland Biotechnology
Institute, Rockville, Maryland 20850; Laboratoire d'Immunologie, Institut de Recherches Cliniques
de Montréal, Montréal, Québec H2W 1R7, Canada; the § Department of Microbiology, Molecular
Biology, and Biochemistry, University of Idaho, Moscow, Idaho 83844; the
Department of
Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455; and the ¶ Basel Institute for Immunology, Postfach CH-4005, Basel, Switzerland
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The three-dimensional structure of the complex between a T cell receptor (TCR) chain
(mouse V
8.2J
2.1C
1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3)
has been recently determined to 3.5 Å resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the
-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we
measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR
chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while
mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic
mice. We show that there is a clear and simple relationship between the affinity of SAGs for
the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or
SEB to the TCR
chain, the greater its ability to stimulate T cells. We also find that there is
an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that
make the greatest energetic contribution to stabilizing the
-SEC3 complex ("hot spot" residues) are strictly conserved among enterotoxins reactive with mouse V
8.2, thereby providing
a basis for understanding why SAGs having other residues at these positions show different V
-binding specificities.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Superantigens (SAGs)1 are a class of disease-causing and
immunostimulatory proteins of bacterial or viral origin.
In addition to causing toxic shock syndrome and food poisoning (1, 2), SAGs have been implicated in a number of
autoimmune disorders, including diabetes mellitus (3),
multiple sclerosis (4), and rheumatoid arthritis (5), through
the activation of T cells specific for self-antigens. SAGs are
able to recognize particular elements on the V domain of
TCRs, largely irrespective of their peptide-MHC specificity, leading to stimulation of a disproportionally large fraction of the T cell population. The activated T cells release
massive amounts of cytokines such as IL-2 and tumor necrosis factor, contributing to the symptoms caused by
SAGs.
The structurally and immunologically best characterized
group of SAGs are the Staphylococcus aureus enterotoxins,
which are mainly associated with food poisoning and toxic
shock syndrome (1, 2). The three-dimensional structure of
the complex between staphylococcal enterotoxin C3
(SEC3) and the chain (V
8.2J
2.1.C
1) of a mouse
TCR (designated 14.3.d) specific for a peptide of influenza virus hemagglutinin (HA 110-120) in the context of I-Ed
shows that CDR2 of the
chain, and to lesser extents
CDR1 and the fourth hypervariable region (HV4), bind in
a cleft between the small and large domains of the SAG (6).
The structure of the TCR
-SEC3 complex agrees well
with mutational and genetic studies implicating residues in
V
CDR1, CDR2, and HV4 in SAG recognition (2, 7).
In addition, mutagenesis of SAGs has revealed that the
stimulatory activity of these molecules is affected when residues at the TCR binding site are altered (8). T cell stimulation by SAGs is generally thought to require simultaneous binding to MHC class II molecules on APCs and the V
element on T cells (9, 10). A model of the TCR-SAG-
MHC complex constructed from the crystal structures of
the TCR-
-SEC3 complex (6), of a TCR V
domain
(11), and of the complex between staphylococcal enterotoxin B (SEB) and an MHC class II molecule (12) suggests that the SAG acts like a wedge between the TCR and
MHC molecules to displace the antigenic peptide away
from the TCR combining site. In this way, the SAG circumvents the normal mechanism for T cell activation by
recognition of specific peptide-MHC complexes (6).
To investigate the relationship between the affinity of
SAGs for TCR and MHC and their ability to activate T
cells, we have measured the binding of a set of SEC3 and
SEB mutants to soluble recombinant 14.3.d chain and to
a human MHC class II molecule, HLA-DR1, loaded with
influenza virus hemagglutinin peptide 306-318 (HA 306-
318). These mutants were generated by alanine-scanning mutagenesis of all SEC3 residues in contact to the TCR
chain in the
-SEC3 crystal structure, or by mutating selected TCR-contacting residues of SEB (which is structurally similar to SEC3 but binds the TCR more weakly) to
those of SEC3. We show that there is a direct correlation
between affinity and mitogenic potency, with SAGs that
have the highest affinity for the TCR
chain being the
most biologically active. We also show that a relatively
small increase in the affinity of the SAG-MHC interaction
is able to compensate a large decrease in SAG-TCR affinity. Finally, a comparison of the so-called "functional
epitope" of SEC3 (those residues that contribute most to
TCR binding) with the "structural epitope" (all SEC3 residues contacting the
chain in the crystal structure) enables us to explain the ability of different SAGs to recognize the same V
elements.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents.
All chemicals were of analytical grade. Restriction endonucleases and DNA modifying enzymes were purchased from New England Biolabs, Inc. (Beverly, MA). Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). Radiolabeled [35S]dATP was from Amersham Corp. (Arlington Heights, IL).Production of Recombinant TCR Chain.
Mutagenesis and Production of SEC3 and SEB Mutants.
The gene for SEB, seb, was obtained from C. Jones and S. Khan (University of Pittsburgh School of Medicine, Pittsburgh, PA; reference 16). Cloning and sequencing of the sec gene encoding SEC3 from Staphylococcal aureus strain FRI913 was reported previously by Hovde et al. (17). All SEB mutants and certain SEC3 mutants were produced by site-directed mutagenesis using a Muta-Gene M13 In Vitro Mutagenesis Kit (Bio-Rad, Richmond, CA). The corresponding genes, on a BamHI/HindIII fragment in the case of SEC3 and on a KpnI/HindIII fragment in the case of SEB, were subcloned into M13mp18. Mutagenic oligonucleotides were designed to replace wild-type codons with ones for alanine in the SEC3 gene or with codons for threonine, tyrosine, or valine in the SEB gene. The mutations were confirmed by DNA sequencing using a Sequenase Version 2.0 Kit (USB, Cleveland, OH). Mutated SEC3 genes were cloned into the expression vector pMIN164 (17) and mutated SEB genes into pUC18. The enterotoxins were expressed in BL21(DE3) Escherichia coli cells, and the periplasmic fraction, in which the SAGs are mostly contained, was obtained by osmotic shock as previously described (18). After dialysis against 20 mM Tris-HCl, pH 7.5, the periplasmic extract was applied to a RedA-Dye column (Amicon, Beverly, MA) as previously described (19). Bound proteins were eluted using a linear 0-500 mM NaCl gradient. The SAG-containing fractions were pooled and concentrated. Alternatively, SEC3 mutants were produced by subcloning the gene for SEC3 on a BamHI/HindIII fragment into pALTER. Oligonucleotide site-directed mutagenesis was accomplished using the Altered Sites In Vitro Mutagenesis Systems Kit (Promega, Madison, WI). Mutations were confirmed by sequencing as above. The mutated genes were cloned into pMIN164 (17) for expression in E. coli DH5Production of Recombinant Peptide-HLA-DR1 Complex.
The pACDR1 virus encoding both soluble DRSedimentation Equilibrium.
Equilibrium measurements of the interactions between the TCR-BIAcore Analysis.
The binding of the TCR-T Cell Proliferation Assay.
4 × 104 lymph node T cells from RAG-2 ![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The functional
contribution of individual SEC3 residues to stabilization of
the complex with the 14.3.d chain was determined by
alanine-scanning mutagenesis of all SEC3 residues in contact to the
chain in the crystal structure (6). Alanine was
chosen because it eliminates the side chain without altering
the main-chain conformation and does not impose any extreme steric or electrostatic effects (24). All affinity measurements were performed using an engineered, unglycosylated version of the 14.3.d
chain because of its stability
and monodisperse behavior in solution. In addition, earlier
studies had shown that this mutant
chain binds to a variety of SAGs with the same Kd as the associated
TCR
heterodimer, or as the glycosylated
chain alone (15).
Binding affinities were determined by sedimentation
equilibrium, a technique with which even weak interactions can be analyzed in solution. Before measuring complex formation between SAGs and the TCR- chain, the
behavior of the individual proteins was assessed by separate
runs. All species behaved well under the conditions used,
with no tendency to aggregate. The calculated molecular weights for the
chain and the enterotoxins deviated <8%
from the expected values based on amino acid composition. To assure the accuracy of our results, we measured
chain-SAG interactions at two different rotor speeds and/
or protein concentrations. The affinity values obtained under these different conditions differed by 30% at most. The
Kds for the interaction of the 14.3.d
chain with a panel of
SEC3 alanine mutants are listed in Table 1, and a representative sedimentation profile is shown in Fig. 1. As can be
seen, the affinities of the various SEC3 mutants for the
chain vary between 3 and 150 µM, with all mutants binding similarly to, or weaker than, the wild-type SAG. Mutation to alanine of three residues (G102, K103, and G106)
within the flexible 93-110 disulfide loop of SEC3 had no
or little effect on binding to the
chain. In contrast, SEC3
mutants N23A, Y90A, and Q210A bind the
chain at
least 50-100-fold less tightly than does the wild-type. At
the protein concentrations used (4-10 µM), the Kds of all
three mutants could be only estimated as ~150 µM, but
the actual values may be significantly greater. Trials using
higher protein concentrations (up to 50 µM) in order to
favor complex formation were unsuccessful due to nonspecific aggregation of the proteins. Mutagenesis of other
SEC3 residues in contact with
chain (T20, Y26, N60,
V91, and F176) resulted in smaller decreases in affinity,
with Kds ranging from 25 to 120 µM.
|
|
We also attempted to obtain mutants of SEC3 and SEB
with higher affinity for the 14.3.d chain in order to see
whether such tighter binding mutants would also show increased ability to stimulate T cells. In the case of the weakly
binding SEB (Kd = 120 µM by sedimentation equilibrium;
Table 1) our approach was to graft residues from the TCR-binding site of SEC3 (Kd = 4.5 µM), which differ between
the two SAGs. As SEB and SEC3 have very similar three-dimensional structures (25, 26) and bind the 14.3.d
chain
in essentially the same orientation (reference 6, Li and Mariuzza, unpublished results), engineering such chimeric SEB
molecules was expected to be straightforward. Excluding
the flexible 93-110 disulfide loop of SEC3 (which, as demonstrated above, does not functionally contribute to binding the
chain), the TCR binding sites of SEC3 and SEB
differ only at positions 20, 26, and 91. We therefore replaced SEB residues L20, V26, and Y91 with SEC3 residues T20, Y26, and V91. We constructed three single mutants (SEB L20T, V26Y, and Y91V) in which these
residues were individually substituted, as well as a triple
mutant in which all three SEC3 residues were grafted onto
the TCR binding site of SEB. As can be seen in Table 1,
only SEB V26Y showed improved affinity: its Kd (as determined by sedimentation equilibrium) was 26 µM, approximately five times lower than that of wild-type SEB, but still
six times higher than that of SEC3. Surprisingly, mutations
L20T and Y91V did not significantly alter the affinity of
SEB for the
chain, whereas no binding whatsoever could
be detected in the case of the the triple mutant.
We also attempted to design mutants of SEC3 with enhanced affinity for the 14.3.d chain on the basis of the
crystal structure of the
-SEC3 complex. For instance,
SEC3 residues Y26 and Y90 were replaced by tryptophan
and V91 by isoleucine with the expectation that the larger
side chains of these residues would make additional van der
Waals contacts with the
chain and contribute to hydrophobic stabilization of the complex. In addition, SEC3 residue G19 was mutated to lysine and F176 to glutamic acid,
since the crystal structure suggested that charged amino acids at these positions could form salt bridges with residues
of opposite charge on the TCR
chain. However, these
mutants either bound the
chain with the same Kd as
wild-type SEC3 (Y26W or V91I), or even displayed up to
10-fold weaker affinities (G19K, Y90W, and F176E; data
not shown).
We coupled most of the SEC3 and SEB mutants
described above directly to the dextran matrix of sensor
chips through the primary amino groups of the proteins.
Unglycosylated 14.3.d chain in increasing concentrations
(1-128 µM) was injected and concentration-dependent surface plasmon resonance profiles such as those shown in
Fig. 2 for SEC3 N60A (panel A) and SEB V26Y (panel B)
were recorded. As in the case of the wild-type proteins, the
binding of all SEC3 and SEB mutants tested was characterized by very fast on- and off-rates. In fact, these kinetic
rates were too fast to accurately measure, although the association and dissociation rate constants may be estimated at
>105 M
1s
1 and >0.1 s
1, respectively. Therefore, affinities were determined under equilibrium binding conditions, in which we took report points for Scatchard analysis
20-40 s after injection. Scatchard plots for SEC3 N60A and
SEB V26Y, after correction for nonspecific binding, are
shown in Fig. 2, C and D. The plots are linear, with correlation coefficients of 0.99; Kds were calculated directly
from the slopes. The predicted maximum specific binding,
calculated from the x-intercept assuming a linear relationship between mass of bound protein and measured RU
(27), indicated that nearly 90% of immobilized SAG molecules retained their binding activity. Similar results were
observed for the other mutants tested; Table 1 lists the Kd
values measured by BIAcore. Comparison of the affinities
obtained by sedimentation equilibrium and BIAcore
showed reasonable agreement, with values for Kd differing
by not more than a factor of two. These variations are
probably attributable to intrinsic differences between the two methods. In the sedimentation equilibrium both binding partners are in solution, whereas in BIAcore one of the
ligands is chemically coupled to a solid support, which may
result in small changes in the conformation and/or accessibility of the immobilized molecule. However, we do not
observe any systematic difference between these two very
different physical methods. In some cases, the affinities obtained from BIAcore were lower (SEC3 T20A, N60A, and
K103A) than those from sedimentation equilibrium, whereas
in others the reverse was true (SEC3 wild-type, Y26A, and
SEB V26Y). Further, we found that both methods gave
very reproducible results, with Kd values deviating by not
more than 30% in each case. We therefore believe that
these two methods combined give an accurate picture of
the functional importance of individual SEC3 or SEB contact residues in complex formation with the TCR-
chain.
|
To mimic normal
physiological conditions as closely as possible, we used resting lymph node T cells bearing the 14.3.d TCR from
RAG-2/
TCR transgenic mice (23) to measure the
stimulatory effects of different SAGs on BALB/c spleen
cells expressing I-Ed (Fig. 3). The results are summarized in
Table 1 as the dose of each SAG required to induce T cell
proliferation 100-fold above background. Dose-response
curves for most of the mutant SAGs did not reach a plateau, even at the highest SAG concentrations tested (Fig.
3). Thus, it is very difficult to estimate either the maximal stimulatory capacity (total proliferation in the assays), or the concentration of SAG that is required to obtain 50% of the
maximal effect. Nevertheless, we believe that our definition of the stimulatory capacity gives an accurate picture of
the relative biological activities of the different SEB and
SEC3 mutants. A comparison with the results from the
binding experiments reveals a direct correlation between
the affinity and the biological activity of SEC3 and SEB
mutants: the higher the affinity of a particular mutant for
the TCR-
chain, the greater its mitogenic potency. For
instance, SEC3 mutants G102A, K103A, and G106A, all
located on the flexible disulfide loop of this SAG, bind to
the
chain with Kds similar to that of wild-type SEC3 and
also show similar biological activity. On the other hand,
SEC3 mutants N23A and Q210A, with Kds
150 µM, exhibit no measurable stimulatory effects whatsoever. However, another very low-affinity mutant, SEC3 Y90A, is still
able to activate T cells, although ~50-fold higher concentrations are needed to achieve the same effect as with wild-type. This difference between no apparent biological activity and low biological activity is reflected in the results
from BIAcore measurements, in which SEC3 Y90A
yielded a Kd of 240 µM, whereas SEC3 N23A and Q210A
did not show any detectable binding (Table 1). Except for F176A, the mutants that have intermediate affinities (i.e.,
SEC3 T20A, Y26A, N60A, V91A) consistently stimulated
T cells transgenic for the 14.3.d TCR less efficiently than
wild-type SEC3, but significantly better than the three mutants with the weakest binding. A very similar picture
emerged from experiments with the various SEB mutants:
SEB V26Y, with its increased affinity, also exhibited a
slightly enhanced mitogenic potency compared to wild-type SEB, whereas the triple mutant, with no detectable
binding to the 14.3.d
chain, only barely stimulated T
cells. However, an apparent exception to this simple affinity-activity rule was the finding that SEB stimulated transgenic T cells ~10-fold better than SEC3, even though the
affinity of SEB for the TCR-
chain is ~35-fold lower
than that of SEC3 (Table 1). This effect was observed using either BALB/c spleen cells or MHC class II-negative
mouse fibroblasts expressing HLA-DR1 as APCs (data not
shown).
|
To determine whether the surprisingly strong mitogenic potency of SEB relative to SEC3 could be attributed to tighter binding to MHC class II on APCs, we examined the binding of SEB and SEC3 to soluble recombinant I-Ed loaded with HA 110-120 (28), as well as to HLA-DR1 loaded with HA 306-318. Both peptide-MHC class II complexes behaved well in separate sedimentation equilibrium runs, which yielded apparent molecular weights of 49 kD for I-Ed and 50 kD for DR1, in close agreement with expected values (52 kD for both I-Ed and DR1). However, we could not detect any binding of I-Ed to either SEB or SEC3, in agreement with BIAcore experiments reported by Kozono et al. using I-Ek (29). In contrast, we could easily measure a Kd of 14 µM for the interaction of SEB with HLA-DR1 and a corresponding value of 48 µM for SEC3. This is also consistent with the observation that DR1 is a much more efficient presenting element for SEB and SEC3 than I-E in T cell stimulation assays (30). Therefore, the unexpectedly high mitogenic potency of SEB relative to SEC3 could be at least partially explained by the tighter binding of SEB to MHC class II. This observation is supported by earlier studies that also suggested a correlation between affinity and biological activity, since SAGs with weaker binding to MHC also showed decreased mitogenic potency (31, 32).
We also determined the affinities of two SEC3 mutants,
G102A and V91A, for HLA-DR1. SEC3 G102A had the
same affinity for the 14.3.d chain as the wild-type SAG,
whereas the affinity of SEC3 V91A was decreased by ~30-fold. In contrast, both mutants bound to HLA-DR1 with
similar affinities (Kd = 54 µM for V91A and Kd = 59 µM
for G102A), and about equally as well as wild-type SEC3. These results demonstrate that mutations in at least these
particular TCR-contacting residues of SEC3 have no influence on affinity for MHC, in agreement with the finding
that the corresponding residues of SEB are not in contact
with MHC in the crystal structure of the SEB-DR1 complex (12).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
While the crystal structure of the 14.3.d chain-SEC3
complex (6) provides detailed information on the molecular architecture of the TCR-SAG interface, it does not tell
us what the actual functional contribution of individual residues to complex stabilization is. To answer this question,
SEC3 residues in contact with the
chain were subjected
to alanine-scanning mutagenesis (24) in order to analyze
their relative importance to binding. The free energy of
binding was calculated from the equation
G =
RT ln
(1/Kd), where R is the univeral gas constant and T is the
absolute temperature. In this way, we constructed a "thermodynamic map" of the TCR binding site of SEC3 that
shows the relative loss of binding-free energy for individual
alanine mutants compared to wild-type SEC3 (
G =
Gmutant
Gwild-type). As shown in Table 1, the most destabilizing alanine substitutions are located at positions
N23, Y90, and Q210 with a
G
2.5 kcal/mol. Significant contributions (1.3 to 2.1 kcal/mol) can also be observed for contact residues T20, Y26, N60, V91, and F176.
In contrast, replacement of G102, K103, and G106 by alanine has little or no effect (<0.5 kcal/mol). In agreement
with this result, these four interface residues form part of
the 93-110 disulfide loop of SEC3 that was only poorly
visible in the crystal structure of the
-SEC3 complex, suggesting high intrinsic mobility (6). Therefore, 8 out of 11 SEC3 contact residues are energetically important in
binding the TCR-
chain such that stabilization of the
-SEC3 complex is achieved by the accumulation of many
productive interactions of varying strength over a large
portion of the interface with the TCR. This finding that a
majority of contact residues significantly contribute to
binding is similar to the case of an anti-hen eggwhite lysozyme antibody binding to an antiidiotypic antibody (22), but different from the binding of human growth hormone
to its receptor, where only a few contact residues appear to
be responsible for the formation of a stable complex (33).
Fig. 4 shows the functional epitope of SEC3 involved in
complex formation with the 14.3.d chain mapped onto
its three-dimensional structure. With the exception of
F176, which lies at the periphery, the most energetically
important SEC3 residues (N23, Y26, Y90, V91, and
Q210) are clustered at the center of the cleft between the
small and large domains of the enterotoxin molecule. All
five of these SEC3 residues interact with a continuous
stretch of amino acids (residues 50-55) in the CDR2 loop
of the V
domain. Hydrophilic residues N23 and Q210
form hydrogen bonds with backbone atoms of this part of
CDR2, whereas hydrophobic residues Y26, Y90, and V91
make a number of van der Waals contacts with main chain
and side chain atoms in the CDR2 loop (6). The finding
that mutations in the highly mobile 93-110 disulfide loop
of SEC3 have little or no effect on binding affinity is in
agreement with the high sequence variability in this region
present in SEC1-3, SEB, and streptococcal pyrogenic exotoxin A (SPEA) (Fig. 5), all of which bind the 14.3.d
chain and activate V
8.2-bearing T cells (15). On the
other hand, "hot spot" residues making the greatest energetic contribution to stabilization of the
-SEC3 complex
(N23, Y90, and Q210), as well as the less important N60,
are strictly conserved in SEC1-3, SEB, and SPEA. Even in
SAGs such as SEA, SED, and SEE, which do not recognize
V
8.2, position 23 is also occupied by an asparagine; this
residue has proven critical for T cell stimulation in earlier
mutagenesis experiments (32). These observations emphasize the functional importance of each of the conserved interface residues, and makes it understandable why SAGs
having other residues at these positions show different V
-binding specificities.
|
|
The results obtained with the SEB-SEC chimeras were
quite surprising, because only SEB mutant V26Y had a
higher affinity (Kd = 19 µM, taken as an average of sedimentation equilibrium and BIAcore measurements) than
wild-type SEB (Kd = 130 µM). The two other SEB single
mutants, L20T and Y91V, did not show improved binding
and the triple mutant did not show any detectable binding to the 14.3.d chain. Since alanine-scanning mutagenesis
of SEC3 implicated Y26 as functionally important, it is reasonable that replacement of the valine that is present in
wild-type SEB by the bulkier tyrosine should increase the
number of van der Waals contacts to the
chain and hence
improve the affinity of that mutant. Other studies also revealed a key role of residue 26 in SEC 1-3 and SEB in the
specificity of these SAGs for different human TCRs. Thus,
SEB and SEC1 react strongly with human V
3 but not
with V
13.1, whereas SEC3 and SEC2 bind well to
V
13.1 but only weakly with V
3 (8). Site-directed mutagenesis has shown that these reactivity differences can be
fully attributed to the amino acid at position 26, which is
valine in SEB and SEC1 but tyrosine in SEC2 and SEC3.
However, it is quite puzzling that the SEB single mutant
Y91V and the SEB triple mutant, with an almost completely grafted SEC3 binding site, react with the 14.3.d
chain only very weakly, or not at all. This behavior could
be explained by unanticipated conformational changes in
SEB induced by these mutations, or by subtle structural differences between the
-SEB and
-SEC3 complexes that
are not apparent at the current resolution of the x ray structures (~3.5 Å). For instance, small differences in the relative orientation of SAG and TCR in the two complexes
may lead to unanticipated effects on binding when interface residues are mutated. Furthermore, attempts to rationally design better-binding SEC3 mutants (e.g., G19K,
Y26W, Y90W, V91I, and F176E) were unsuccessful, since
in all cases the affinity was the same as that of wild-type
SEC3, or even up to 10-fold lower. It is therefore entirely
possible that the TCR binding site of SEC3 is already optimized and that the introduction of charged (G19K, F176E)
or bulkier (Y90W) residues leads to less favorable interactions and a consequent decrease in affinity.
Our comparison between affinity and biological activity
clearly shows that SAG mutants that bind the TCR-
chain more tightly stimulate T cells more efficiently than
do SAG mutants with lower affinities. This observation
complements recent studies of T cell activation by peptide-
MHC ligands, which strongly suggest that the affinity of
the TCR-peptide-MHC interaction plays a critical role in
determining the nature of the T cell response (34). For example, it has been shown that peptide-MHC antagonists
that induce T cell anergy may act by binding the TCR
with affinities that are below those of peptide-MHC agonists. Furthermore, the so-called "serial triggering" model
of T cell activation argues that the rather low affinities and
fast off-rates observed for many peptide-MHC-TCR interactions are in fact necessary to enable a single peptide-
MHC complex to serially engage a large number of TCRs
(35, 36). Indeed, ligands with too high of an affinity for the
TCR are predicted to be actually less efficient at triggering T cells. We have shown that, like peptide-MHC, the
binding of SAGs to the TCR is characterized by relatively
low affinities and fast dissociation rates. This suggests that
SAGs "mimic" the interaction of peptide-MHC complexes with the TCR in terms of affinities and kinetics, a
conclusion also drawn by Viola and Lanzavecchia (36).
However, it remains to be established whether there is indeed an optimum affinity for T cell activation by SAGs, such that SAG variants with either higher or lower affinities than this optimum value exhibit decreased ability to stimulate T cells. Alternatively, mutant SAGs with progressively
higher affinities for the TCR relative to the wild-type
would stimulate T cells increasingly well, until some plateau of maximum stimulation is reached. Unfortunately,
our current data do not permit us to distinguish between
the two possibilities since we were unable to engineer SAG
variants with markedly enhanced binding to the TCR-
chain. In the best case, SEB V26Y bound to the
chain
only approximately sevenfold more tightly than did the
wild-type SAG.
The only notable discrepancy in the observed correlation
of activity with affinity is that SEB exhibited an ~10-fold
higher potency than SEC3 in T cell stimulation assays,
even though its affinity for the TCR- chain (Kd = 130 µM) is much lower than that of SEC3 (Kd = 3.8 µM).
Since the activation of T cells by SAGs requires simultaneous binding of the SAG to TCR and to MHC on the
APCs, this discrepancy could be reasonably explained by a
tighter binding of SEB than SEC3 to MHC molecules.
The crystal structure of the complex between HLA-DR1
and SEB (12) reveals that MHC-contacting residues of SEB
are different in SEC3 (Fig. 5). These sequence differences
may well result in different affinities of SEB and SEC3 for
MHC class II and thereby exert a significant effect on the
mitogenic potency of these SAGs. Indeed, we found that
SEB binds to HLA-DR1 with a Kd of 14 µM, whereas the
corresponding value for SEC3 was 48 µM. This threefold
higher affinity of SEB for MHC appears to compensate for
its 35-fold lower affinity for the TCR. However, it is
somewhat puzzling that this rather modest difference in
SAG-MHC affinity has such a large impact on biological activity, especially given that the effects of changes in
TCR-SAG affinity on SAG activity are proportionately
much less pronounced (Table 1). One explanation may be
that the tighter binding of SEB to HLA-DR1 is mainly the
result of a slower off-rate leading to a considerably longer
residence time of SEB molecules on the MHC and therefore to much more efficient presentation of the SAG to the
T cell. Another could be that some minor structural differences between the trimolecular MHC-SEB-TCR and
MHC-SEC3-TCR complexes may result in altered binding properties that cannot be easily discerned by examining
either the MHC-SAG or TCR-SAG complexes individually. Another important question regarding T cell activation by SAGs remains as well: how can a SAG efficiently
cross-link the APC and T cell if its affinity for both MHC
and TCR is quite weak? This would appear to be less of an
issue for conventional T cell activation by peptide-MHC
complexes because only one low-affinity interaction is involved. One possibility is that accessory molecules such as
CD4 help stabilize the TCR-SAG-MHC complex sufficiently for activation to occur. Another is that the overall
stability of the MHC-SAG-TCR complex is greater than
one would expect from considering the TCR-SAG and
MHC-SAG interactions independently. In other words,
the binding of SAGs to TCR and MHC may be a cooperative process such that the SAG-MHC complex binds the
TCR with a higher affinity than does SAG alone. Indeed,
surface plasmon resonance experiments have suggested a
tighter binding of an immobilized TCR to SEB complexed
with HLA-DR1 than to SEB alone (37). Furthermore, our
current model of the MHC-SAG-TCR complex (6) suggests that the V
domain of the TCR can contact the
MHC
1 domain, perhaps helping to stabilize the overall
complex. However, only an actual crystal structure determination of an MHC-SAG-TCR complex, combined with further binding experiments with all three components, will reveal whether cooperative effects in the trimolecular complex in fact occur, and if so, how exactly they
act to determine T cell stimulation by SAGs.
![]() |
Footnotes |
---|
Address correspondence to Dr. Roy A. Mariuzza, Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Dr., Rockville, MD 20850. Phone: 301-738-6243; Fax: 301-738-6255; E-mail: mariuzza{at}indigo2.carb.nist.gov
Received for publication 18 September 1997 and in revised form 22 December 1997.
This research was supported by National Institutes of Health (NIH) grant 36900 and National Multiple Sclerosis Society grant RG2747 (R.A. Mariuzza); NIH grant HL-36611 (P.M. Schlievert); NIH grant AI-28401 and USDA grant 9402399 (G.A. Bohach); and grants from the National Cancer Institute of Canada (R.-P. Sékaly). Support from the Lucille P. Markey Charitable Trust is also gratefully acknowledged. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-LaRoche Ltd., Basel, Switzerland. L. Leder is a Fellow of the Swiss National Science Foundation. R.-P. Sékaly holds a Medical Research Council of Canada Scientist Award.We thank Claudia Beck for technical assistance and Cynthia V. Stauffacher (Purdue University, West Lafayette, IN) for providing coordinates of SEC3.
Abbreviations used in this paper HA, hemaglutinin peptide; HBS, Hepes-buffered saline; Kd, dissociation constant; RU, resonance unit; SAG, superantigen; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; SEC, staphylococcal enterotoxin C; SED, staphylococcal enterotoxin D; SEE, staphylococcal enterotoxin E; SPEA, streptococcal pyrogenic exotoxin A.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bohach, G.A., D.J. Fast, R.D. Nelson, and P.M. Schlievert. 1990. Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses. Crit. Rev. Microbiol. 17: 251-272 [Medline]. |
2. | Kotzin, B.L., D.Y.M. Leung, J. Kappler, and P. Marrack. 1993. Superantigens and their potential role in human disease. Adv. Immunol. 54: 99-106 [Medline]. |
3. | Conrad, B., R.N. Weissmahr, J. Boni, R. Arcari, J. Schupbach, and B. Mach. 1997. A human endogeneous retroviral superantigen as candidate autoimmune gene in type I diabetes. Cell. 90: 303-313 [Medline]. |
4. | Brocke, S., A. Gaur, C. Piercy, A. Gautam, K. Gijebls, C.G. Fathman, and L. Steinman. 1993. Induction of relapsing paralysis in experimental autoimmune encephalomyelitis by bacterial superantigen. Nature. 365: 642-644 [Medline]. |
5. | Cole, B.C., and M.M. Griffiths. 1993. Triggering and exacerbation of autoimmune arthritis by the mycoplasma arthritidis superantigen MAM. Arthritis Rheum. 36: 994-1002 [Medline]. |
6. |
Fields, B.A.,
E.L. Malchiodi,
H. Li,
X. Ysern,
C.V. Stauffacher,
P.M. Schlievert,
K. Karajalainen, and
R.A. Mariuzza.
1996.
Crystal structure of a T cell receptor ![]() |
7. |
Patten, P.A.,
E.P. Rock,
T. Sonoda,
B. Fazekas de St,
Groth,
J.L. Jorgensen, and
M.M. Davis.
1993.
Transfer of putative
complementarity-determining region loops of T cell receptor
V domains confers toxin reactivity but not peptide/MHC specificity.
J. Immunol.
150:
2281-2294
|
8. | Deringer, J.R., R.J. Ely, C.V. Stauffacher, and G.A. Bohach. 1996. Subtype-specific interactions of type C staphylococcal enterotoxins with the T-cell receptor. Mol. Microbiol. 22: 523-534 [Medline]. |
9. | Woodland, D.L., and M.A. Blackman. 1993. How do T cell receptors, MHC molecules and superantigens get together? Immunol. Today. 14: 208-212 [Medline]. |
10. | Webb, S.R., and N.R.J. Gascoigne. 1994. T-cell activation by superantigens. Curr. Opin. Immunol. 6: 467-475 [Medline]. |
11. |
Fields, B.A.,
E.L. Ober,
M.I. Lebedeva,
B.C. Braden,
X. Ysern,
J.K. Kim,
X. Shao,
E.S. Ward, and
R.A. Mariuzza.
1995.
Crystal structure of the V![]() |
12. | Jardetzky, T.S., J.H. Brown, J.C. Gorga, L.J. Stern, R.G. Urban, Y. Chi, C.V. Stauffacher, J.L. Strominger, and D.C. Wiley. 1994. Three-dimensional structure of a human class II histocompability antigen complexed with superantigen. Nature. 368: 711-718 [Medline]. |
13. |
Bentley, G.A.,
G. Boulot,
K. Karjalainen, and
R.A. Mariuzza.
1995.
Crystal structure of the ![]() |
14. |
Kubo, R.T.,
W. Born,
J.W. Kappler,
P. Marrack, and
M. Pigeon.
1989.
Characterization of a monoclonal antibody
which detects all murine ![]() ![]() |
15. |
Malchiodi, E.L.,
E. Eisenstein,
B.A. Fields,
D.H. Ohlendorf,
P.M. Schlievert,
K. Karajalainen, and
R.A. Mariuzza.
1995.
Superantigen binding to a T cell receptor ![]() |
16. | Jones, C.L., and S.A. Kahn. 1986. Nucleotide sequence of the enterotoxin B gene from Staphylococcus aureus. J. Bacteriol. 166: 29-33 [Medline]. |
17. | Hovde, C.J., S.P. Hackett, and G.A. Bohach. 1990. Nucleotide sequence of the staphylococcal enterotoxin C3: sequence comparison of all three type C staphyloccal enterotoxins. Mol. Gen. Genet. 220: 329-333 [Medline]. |
18. | Ward, E.S.. 1992. Secretion of T-cell receptor fragments from recombinant Escherichia coli cells. J. Mol. Biol. 224: 885-888 [Medline]. |
19. | Passalacqua, E.F., R.D. Brehm, K.R. Acharya, and H.S. Tranter. 1993. Crystallization and preliminary X-ray analysis of a microbial superantigen staphylococcal enterotoxin C2. J. Mol. Biol. 233: 170-172 [Medline]. |
20. | Summers, M.D., and G.E. Smith. 1988. A manual of methods for baculovirus and insect cell culture procedures. In Texas Agricultural Experiment Station Bulletin No. 1555. College Station, TX. |
21. |
Stern, L.J., and
D.C. Wiley.
1992.
The human class II MHC
protein HLA-DR1 assembles as empty ![]() ![]() |
22. | Dall'Aqua, W., E.R. Goldman, E. Eisenstein, and R.A. Mariuzza. 1996. A mutational analysis of the binding of two different proteins to the same antibody. Biochemistry. 35: 9667-9776 [Medline]. |
23. | Kirberg, J., A. Baron, S. Jakob, A. Rolink, K. Karjalainen, and H. von Boehmer. 1994. Thymic selection of CD8+ single positive cells with a class II major histocompatibility complex-restricted receptor. J. Exp. Med. 180: 25-34 [Abstract]. |
24. | Wells, J.A.. 1991. Systematic mutational analysis of protein-protein interfaces. Methods Enzymol. 202: 390-411 [Medline]. |
25. | Swaminathan, S., W. Furey, J. Pletcher, and M. Sax. 1992. Crystal structure of staphylococcal entertoxin B, a superantigen. Nature. 359: 801-806 [Medline]. |
26. | Schlievert, P.M., G.A. Bohach, D.H. Ohlendorf, C.V. Stauffacher, D.Y. Leung, D.L. Murray, G.S. Prassad, C.A. Earhart, L.M. Jablonski, M.L. Hoffmann, and Y.I. Chi. 1995. Molecular structure of staphylococcus and streptococcus superantigens. J. Clin. Immunol. 15: 4-10 . |
27. | Granzow, R., and R. Reed. 1992. Interactions in the fourth dimension. Biotechnology. 10: 390-393 [Medline]. |
28. | Wallny, H.J., G. Sollami, and K. Karjalainen. 1995. Soluble major histocompatibility complex class II molecules produced in Drosophila cells. Eur. J. Immunol. 25: 1262-1266 [Medline]. |
29. | Kozono, H., D. Parker, J. White, P. Marrack, and J. Kappler. 1995. Multiple binding sites for bacterial superantigens on soluble class II MHC molecules. Immunity. 3: 187-196 [Medline]. |
30. | Herman, A., G. Croteau, R.P. Sékaly, J. Kappler, and P. Marrack. 1990. HLA-DR alleles differ in their ability to present staphyloccal enterotoxins to T cells. J. Exp. Med. 172: 709-717 [Abstract]. |
31. |
Chintagumpala, M.M.,
J.A. Mollick, and
R.R. Rich.
1991.
Staphylococcal toxins bind to different sites on HLA-DR.
J. Immunol.
147:
3876-3881
|
32. | Kappler, J.W., A. Herman, J. Clements, and P. Marrack. 1992. Mutations defining functional regions of the superantigen staphylococcal enterotoxin B. J. Exp. Med. 175: 387-396 [Abstract]. |
33. | Cunningham, B.C., and J.A. Wells. 1993. Comparison of a structural and functional epitope. J. Mol. Biol. 234: 554-563 [Medline]. |
34. | Sloan-Lancaster, J., and P.M. Allen. 1996. Altered peptide-ligand induced partial T cell activation: molecular mechanisms and role in T cell biology. Annu. Rev. Immunol. 14: 1-27 [Medline]. |
35. | Valitutti, S., S. Muller, M. Cella, E. Padovan, and A. Lanzavecchia. 1995. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature. 375: 148-151 [Medline]. |
36. | Viola, A., and A. Lanzavecchia. 1996. T cell activation determined by number and tunable thresholds. Science. 273: 104-106 [Abstract]. |
37. | Seth, A., L.J. Stern, T.H.M. Ottenhoff, I. Engel, M.J. Owen, J.R. Lamb, R.D. Klausner, and D.C. Wiley. 1994. Binary and ternary complexes between T cell receptor, class II MHC and superantigen in vitro. Nature. 369: 324-327 [Medline]. |