Zinc Binding and Dimerization of Streptococcus
pyogenes Pyrogenic Exotoxin C Are Not Essential for T-cell
Stimulation*
Wieslaw
Swietnicki
,
Anne M.
Barnie,
Beverly K.
Dyas, and
Robert
G.
Ulrich§
From the United States Army Medical Research Institute of
Infectious Diseases, Frederick, Maryland 21702
Received for publication, July 11, 2002, and in revised form, December 4, 2002
 |
ABSTRACT |
Streptococcal pyrogenic enterotoxin C
(Spe-C) is a superantigen virulence factor produced by
Streptococcus pyogenes that activates T-cells polyclonally.
The biologically active form of Spe-C is thought to be a homodimer
containing an essential zinc coordination site on each subunit,
consisting of the residues His167, His201, and
Asp203. Crystallographic data suggested that
receptor specificity is dependent on contacts between the zinc
coordination site of Spe-C and the
-chain of the major
histocompatibility complex type II (MHCII) molecule. Our results
indicate that only a minor fraction of dimer is present at T-cell
stimulatory concentrations of Spe-C following mutation of the unpaired
side chain of cysteine at residue 27 to serine. Mutations of amino acid
residues His167, His201, or Asp203
had only minor effects on protein stability but resulted in greatly diminished MHCII binding, as measured by surface plasmon resonance with
isolated receptor/ligand pairs and flow cytometry with MHCII-expressing cells. However, with the exception of the mutants D203A and D203N, mutation of the zinc-binding site of Spe-C did not significantly impact
T-cell activation. The mutation Y76A, located in a polar pocket
conserved among most superantigens, resulted in significant loss of
T-cell stimulation, although no effect was observed on the overall
binding to human MHCII molecules, perhaps because of the masking of
this lower affinity interaction by the dominant zinc-dependent binding. To a lesser extent, mutations of
side chains found in a second conserved MHCII
-chain-binding site consisting of a hydrophobic surface loop decreased T-cell stimulation. Our results demonstrate that dimerization and zinc coordination are not essential for biological activity of Spe-C and suggest the
contribution of an alternative MHCII binding mode to T-cell activation.
 |
INTRODUCTION |
The 24.5-kDa Spe-C1
protein is an exotoxin produced by the pathogenic bacterium
Streptococcus pyogenes. Together with staphylococcal enterotoxin B (SEB), SEC, toxic shock syndrome toxin (TSST), and several others, Spe-C belongs to the superantigen protein family. Superantigens help pathogenic bacteria colonize the host by binding to
major histocompatibility complex class II (MHCII) and T-cell receptors
(TCR), thus bypassing the normal signal transduction pathway essential
for immune recognition. In addition, the resulting nonspecific
stimulation of the host's T-cells can induce pathological levels of
cytokines. There is a substantial amount of structural data available
for the superantigens, both free and in complex with their TCR and
MHCII receptors (1-6). The superantigen fold is highly conserved
despite low overall sequence similarity among protein family members.
Superantigen-binding sites are found on the
-chain or on the
-chain of the MHCII receptor. The
-chain-binding site requires
zinc to form a complex with the superantigen through a conserved His
from MHCII and three His or Asp residues from the superantigen. The
absence of zinc in certain superantigens, such as SEB, presumably
precludes
-chain binding. The
-chain-binding site requires a
structurally conserved positioning of a hydrophobic binding loop
contributed by the superantigen, and usually a second polar binding
pocket is also engaged. For the case of Spe-C, the
-chain binding
loop is displaced, and the potential
-chain-binding site, if the
Spe-C were to bind MHCII as a monomer, may be hidden in the interface
of the zinc-dependent superantigen dimer (2), hypothetically resulting in interaction only with the MHCII
-chain. The
-chain binding residues are conserved in Spe-C and other streptococcal superantigens, and the
-chain-binding site (3, 4) is
intact in crystallographic models in which this interaction site with
MHCII critically involves zinc. Zinc was implicated in streptococcal
superantigen binding to MHCII on the surface of cells (7-9),
suggesting an essential role in both MHCII molecular recognition and
TCR-mediated signal transduction. The high affinity zinc coordination
complexes of SEA and Spe-C consist of three amino acid side chains
contributed by the superantigen and the fourth by the MHCII receptor.
As observed in structural data for the Spe-C tetrahedral zinc
coordination complex (3-5), His and/or Asp contribute the zinc ligands
from the superantigen and His81 from the
-chain of MHCII
molecule. The recently determined crystal structure of SEH with the
MHCII HLA-DR1 indicates a potentially lower affinity zinc
coordination site, present at the interface with the receptor (6),
where one of the ligands is a water molecule. The binding of SEH to
MHCII is dependent on zinc, and superantigen potency is diminished when
the zinc binding residues are replaced by alanines (10). However, the
MHCII
-chain mutation H81A, expected to abolish the zinc-mediated
SEH binding to MHCII, had minimal effect (10). In addition, the
C-terminal zinc-binding site of another dimer-forming superantigen
(11), SEC1, is not necessary for T-cell stimulation (12). In
vitro data with purified recombinant Spe-C demonstrated a large
proportion of dimer at 81 µM protein concentration (7),
yet the Kd for dimer formation from equilibrium
centrifugation data is only 390 µM (3) compared with
T-cell stimulation occurring at nanomolar concentrations of
superantigen. The mutant H35A, thought to affect dimer formation, had
only minor effect on the Kd (3). Finally, the Spe-C
molecule forms a zinc-less dimer in the Spe-C/TCR V
2.1 crystal
structure (13), using a surface proposed to be involved in the
zinc-mediated MHCII binding, a site far removed from the one
anticipated to bury the conserved residues interacting with the MHCII
-chain (2). Therefore, to further understand binding of Spe-C to
MHCII and the contribution to T-cell stimulation, we have re-examined
the relationship of zinc binding to stability, oligomerization, and
biological function of the Spe-C superantigen. Our results suggest a
diminished functional role for zinc and dimerization in T-cell
stimulation. Further, an alternative zinc-independent-binding site,
involving residues structurally equivalent to the MHCII
-chain-binding hydrophobic loop and polar pocket on SEB, may be
biologically relevant for TCR stimulation. Binding to this site is only
possible when the biologically active form of Spe-C is a monomer
but not the homodimer.
 |
MATERIALS AND METHODS |
cDNA Cloning and Plasmid Construction--
Genomic DNA was
purified (Wizard Genomic DNA Isolation Kit; Promega) from a
Spe-C+ clinical isolate of S. pyogenes grown in
a culture. The cDNA corresponding to the wt Spe-C was amplified by
PCR and cloned into pRSET A vector between NheI and
HindIII sites, together with a linker coding for a thrombin
cleavage site (Leu-Val-Pro-Arg*Gly-Ser) at the N
terminus of Spe-C. The final construct coded for a fusion protein of
His6 (pRSET A vector) followed by a thrombin linker attached to the N terminus of mature Spe-C (amino acids 28-235). The
protein construct was designed to have a Gly-Ser N-terminal extension
after thrombin cleavage. To avoid covalent dimer formation because of
an intermolecular disulfide bridge, a C27S mutation was introduced by
site-directed mutagenesis (QuikChange; Stratagene). The full open
reading frame corresponding to the fusion protein was sequenced
on a CEQ 2000XL (Beckman, Fullerton, CA) sequencer. For protein
expression, the plasmid DNA was transformed into a BL21 (DE3) strain (Invitrogen).
Protein Expression and Purification--
A single colony from a
freshly streaked LB + Amp (100 µg/ml) plate was grown in a 3 ml of LB + Amp (100 µg/ml) medium at 37 °C in a shaker for ~4 h until it
became visibly turbid. A 0.5-ml aliquot was used to inoculate 25 ml of
LB + Amp (100 µg/ml) medium and grown for an additional 4-5 h. The
culture was then stored for 12 h at 4 °C. The next day, a 6-ml
aliquot was added to 500 ml of LB + Amp (100 µg/ml) medium, and the
culture was grown in a shaker at 37 °C until it reached
A600 of 1.0. At that point, isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 1 mM to induce protein expression,
and the culture was grown overnight at 37 °C. Cells were harvested
by centrifugation and stored at
80 °C until further processing.
To purify protein, bacterial cells from a 2-liter culture were thawed
at room temperature and resuspended in ~120 ml of denaturing buffer
(6 M GdnHCl, 100 mM potassium phosphate,
10 mM Tris-HCl, pH 8.0). Cells were disrupted by sonication
on ice (3 × 1 min with a 1-min rest) using a Model 300 sonic
dismembrator (Fisher Scientific, Hampton, NH) equipped with a
1/4-in-diameter probe. Cellular debris was removed by
centrifugation (2 h, 20,000 × g, 4 °C), and the
supernatant was mixed with 25 ml of nickel-nitrilotriacetic acid
Superflow resin (Qiagen) for 30 min to bind the protein. The resin
suspension was used to pack a XK 26/20 column (Amersham Biosciences) in
a batch mode, and the column was connected to a ÄKTA fast protein
liquid chromatography system (Amersham Biosciences). Most cellular
impurities were removed by washing with the denaturing buffer, and the
protein was refolded by running a linear gradient of 100-0% of
denaturing buffer and 100 mM potassium phosphate, 10 mM Tris-HCl, pH 8.0, over a 2-h time at a flow rate of 1 ml/min. Residual impurities were removed by a wash with 50 mM imidazole, 100 mM potassium phosphate, 10 mM Tris-HCl, pH 8.0, and the protein was eluted with 500 mM imidazole, 100 mM potassium phosphate, pH
5.8. The fractions containing protein, as judged by an SDS-PAGE analysis, were pooled and dialyzed against 10 mM Tris-HCl,
1 mM EDTA, pH 8.0. The His6-thrombin linker was
removed by digestion (20 °C, 7 d) with 10 units of thrombin
(Amersham Biosciences) per mg of protein. The residual thrombin was
removed by incubation with 1 ml of Q-Sepharose (Amersham Biosciences)
resin, and the supernatant was dialyzed against 10 mM
potassium phosphate, 1 mM EDTA, pH 5.8. The protein was
bound to a SP-Sepharose (Amersham Biosciences) resin in a batch mode,
and the suspension was used to pack a XK 26/20 column (Amersham
Biosciences). The column was washed with 10 mM potassium
phosphate, 1 mM EDTA, pH 5.8, buffer and developed with a
linear gradient of 0-0.5 M NaCl at a 5 ml/min flow rate. Fractions
containing protein, as judged by a SDS-PAGE analysis, were pooled and
dialyzed against 10 mM HEPES, pH 7.0. The protein was
concentrated in a Centriplus-3 (Millipore, Bedford, MA) concentrator,
aliquoted, and stored at
80 °C. Protein concentration was
determined by UV using molar extinction coefficients calculated with a
ProtParam program on the ExPASy proteomics server at the Swiss
Institute of Bioinformatics (Genève, Switzerland). Typically, 10-15 mg of purified protein at 0.5-2.5 mg/ml was obtained from 2L of
bacterial cell culture. The purity of protein was verified by a
combination of a SDS-PAGE and reversed-phase liquid chromatography. Proteins were characterized by LC-MS and, if needed, by an N-terminal amino acid sequencing. The final purity was greater than 95% as judged
by LC-MS. The recombinant HLA-DR1 protein was prepared as
described (24). The protein was stored in 20% glycerol at
20 °C at 5 mg/ml. A fresh aliquot was removed from the freezer and
placed on ice directly before the experiments.
Mutant Construction and Purification--
Mutants were
constructed by site-directed mutagenesis (QuikChange; Stratagene). The
full open reading frames corresponding to Spe-C fusion proteins were
sequenced on CEQ 2000XL DNA sequencer (Beckman), and the plasmids were
transformed into an Escherichia coli BL21 (DE3) strain for
protein expression. Bacterial cell growth and protein purification were
identical to the r wt protein (above). All mutant proteins contained
the C27S replacement.
Metal Content Analysis--
A protein sample volume
corresponding to 0.5-1.0 mg of total protein was transferred to an
empty sample container, weighed, and diluted to 5 ml with 2% nitric
acid. The diluted sample was then analyzed by Hewlett Packard 4500 series inductively coupled plasma spectroscopy for zinc and
nickel content. Detection limits for the method were 1 µg of metal
per liter of solution.
LC-MS Analysis--
The analysis was performed on a Finnigan LCQ
Deca LC-MS. Typically, a 15-µl aliquot of protein solution was
injected on a reversed-phase Poros II R/H 8 × 100-mm column (LC
Packings, San Francisco, CA) equilibrated in 0.1% formic acid. The
column was developed with a linear 0-100% gradient of 0.1% formic
acid/80% acetonitrile over a period of 30 min at the flow rate of
0.250 ml/min. Eluent was also monitored at A280
on Agilent 1100 series UV-visible detector (Agilent Technologies, Palo
Alto, CA). The data were stored on a computer and processed with the
instrument's software.
Size Exclusion Chromatography--
A total of 250 µl of
protein solution at a concentration of 0.2 mg/ml in running buffer (100 mM potassium phosphate, 50 mM NaCl, pH 7.0) was
injected onto Superdex 200 HR 10/30 (Amersham Biosciences) gel
filtration column connected to a ÄKTA fast protein liquid
chromatography system (Amersham Biosciences). Protein separation at the
flow rate of 0.5 ml/min was monitored by following
A280 of the eluent. The column was calibrated
with gel chromatography markers (Amersham Biosciences). Molecular
weight calculations of mutant Spe-C proteins were determined from plots
of the logarithm of molecular weight of standards versus
retention volume. All calculations assumed a globular shape of proteins.
Circular Dichroism Measurements--
All measurements were
made on a J-810 spectropolarimeter (Jasco, Inc., Easton, MD) equipped
with a Peltier unit to control temperature of sample holding block.
Unless otherwise specified, the block temperature was maintained at
25 °C. Far-UV spectra were collected in a 1-mm path length
rectangular cuvette. Typically, at 0.2 mg/ml protein solution in 50 mM potassium phosphate, pH 7.0, data from ten spectra were
collected and averaged. The spectra were corrected for the buffer,
smoothed with programs included with the Jasco software, and converted
to mean residue ellipticity, MRE, according to Equation 1,
where
is the measured ellipticity in millidegree,
c the protein concentration in mol/liter, d is the path length in cm, and Na is the number of
amino acid residues per molecule.
|
(Eq. 1)
|
Secondary structure estimates were performed on the
buffer-corrected, unconverted data with a Neural Network program from the Softsec program suite (Softwood Software).
Equilibrium unfolding experiments were performed with the Jasco
titrator unit equipped with two 2.5-ml Hamilton syringes. Typically,
about 10 ml of protein solution with concentration of 20 µg/ml in
GdnHCl (5.4-5.6 M), 50 mM potassium phosphate, pH
7.0, was prepared, and about 2 ml was added in 50-µl increments to
2.7 ml of identical solution without GdnHCl in a 1-cm path length
cuvette with constant mixing. The instrument and titrator parameters
were as follows: titration steps, 40; mixing time after denaturant injection, 150 s; equilibration after solution
withdrawal, 10 s; wavelength, 222 nm; response time, 8 s;
average, two times; and bandwidth, 2 nm. Under these conditions, the
signal recovery for refolding reaction was at least 90%. Data from
unfolding were converted to ellipticity versus denaturant
concentration by the Jasco software, transferred to Kaleidagraph
program (Synergy Software, Reading, PA), and used for a non-linear
fitting according to Equation 2, where
222 is
the mean residue ellipticity at 222 nm, a is the slope, and
b is the intercept of
versus
denaturant concentration of the native, N, and unfolded,
U, states, R is the gas constant equal to 8.315 J/mol M deg K, T is the temperature in K, m is the slope of
G versus denaturant
concentration, c, and Cm is the
concentration of denaturant at which 50% of the protein is unfolded.
|
(Eq. 2)
|
The free energy of unfolding at denaturant concentration equal
to zero,
Go, was calculated according to
Equation 3.
|
(Eq. 3)
|
All measurements were made in duplicate or triplicate. Data are
reported as means ± S.D. between independent measurements.
T-cell Proliferation Assay--
Isolated human blood mononuclear
cells were cultured for three days in 96-well plates (3-5 × 105 cells/well) in medium containing 5% fetal bovine serum
(Invitrogen) and pulsed with 1 µCi of
[3H]thymidine (Amersham Biosciences) for a period of
9 h. SEA, SEB, and Spe-A were obtained from Toxin Technology, Inc.
(Sarasota, FL). Cells were incubated in triplicate with bacterial
toxins or recombinant Spe-C mutants and disrupted osmotically, and
cellular debris was transferred to counting cassettes. Radioactivity
was measured on a liquid scintillation counter (TopCount-NXT; Packard Instruments).
Endotoxin Level Determination--
Endotoxin level measurements
were measured by a Limulus-lysate assay (QCL-1000; BioWhittaker,
Walkersville, MD). All protein samples had levels less than 0.24 endotoxin units/liter of solution.
Interactions of MHCII and Superantigens--
Surface plasmon
resonance measurements of ligand-receptor binding were performed on a
Biacore 3000 (Biacore Inc., Piscataway, NJ). In a typical
experiment, a solution of recombinant HLA-DR1 in 10 mM
HEPES, pH 7.0, 150 mM NaCl was passed over a CM5 chip surface with immobilized superantigen (about 3000 refractive unit) at
20 µl/min for 3 min at 37 °C. The dissociation phase was followed for 2 min at the same flow rate, and the surface was regenerated with
10 mM EDTA and 2 M KCl. The sensogram was
always corrected on a reference signal originating from a
non-derivatized surface, measured in the same experiment. All data were
processed for best fit using software supplied by the manufacturer and
assuming a simple 1:1 Langmuire association model for the on-rate,
kon, off-rate, koff, and dissociation constant,
Kd, calculations. A cell-based MHCII
binding assay was also used. The human B-lymphoblastoid cell line LG2
(16) was incubated with FITC-labeled r wt in Hanks' basic salt
solution medium supplemented with 0.1% bovine serum albumin for 30 min
at 37 °C. The cells were then washed with the medium, fixed with 1%
paraformaldehyde in phosphate-buffered saline, and analyzed by a laser
fluorescence-activated flow cytometry (BD Biosciences). Alternatively,
mouse L cells expressing human DR
/DR1
(*B0101), prepared as
previously described (11), were grown to 80% confluency. The cells
were removed from tissue culture flasks with 25 mM EDTA in
HBSS calcium- and magnesium-free medium, washed with Eagle's minimal
essential medium supplemented with non-essential amino acids, and
incubated with unlabeled r wt at the concentration range of
0.16-10 µM for 30 min at 37 °C.
FITC-labeled r wt was then added to a final concentration of 1.25 µM, and the mixture was incubated for an additional 30 min at 37 °C. The cells were washed with Eagle's minimal essential
medium, fixed with 1% paraformaldehyde in phosphate-buffered saline,
and analyzed by laser fluorescence-activated flow cytometry (BD Biosciences).
Homology Modeling--
Protein sequences were downloaded from a
public data base through the PubMed program (National Library of
Medicine). All models were constructed by Swiss Model program (26) on
the ExPASy server at the Swiss Institute of Bioinformatics
(Genève, Switzerland). The models optimized by the server and
examined with the WHAT IF program (27) were either corrected manually
or discarded if errors were too large. The crystallographic structure
of SEH in complex with HLA-DR1 (6) was used to validate the
homology modeling method. There was a high degree of correlation
between modeled and experimental structures, presenting root mean
square deviation of 0.4 and 1.0 Å for the
-C backbone of the
superantigen alone and the whole complex, respectively.
Protein Docking--
Molecular models were built with the
Flexidock program in the Biopolymer module of Sybyl 6.7 (Tripos
Software, St. Louis, MO) molecular modeling program suite. Starting
templates were constructed by overlaying the
-C backbones of Spe-C
on SEB from the SEB/DR1 complex (19). The Flexidock program uses a
variation of a genetic algorithm (27) that employs a combination of
rigid body and torsional space search. Final solutions are scored by the fitness function, which includes van der Waals, electrostatic, and
torsional energy terms of the default Tripos force field, with the
following modifications: hydrogen van der Waals radius, 1 Å; hydrogen
bond epsilon, 0.03; and van der Waals cutoff distance for centroids, 16 Å. The strategy was used successfully for docking other proteins or
small ligands (see Refs. 28-31). Because of the random nature of the
genetic algorithm's initial search, we repeated all docking
calculations for Spe-C. All solutions were the same as in the first
simulation. Therefore, the structures presented represent converged,
calculated structures. A typical run for docking of Spe-C or other
superantigens to DR1 used default parameter settings with a 10-Å
search radius around the starting binding pocket to maximize the search
space. Contributions from electrostatics and all hydrogens were always
included in the search parameters. A default initial seed number was
used, but the total number of generations of Flexidock search was
always limited to 3000 to avoid bias toward the best scoring solution.
The following two approaches were selected for docking of Spe-C to DR1:
1) rigid body docking without flexing side chains; 2) all side chains
of ligand and receptor were considered adjustable. Although the first strategy resulted in many solutions, all but one was considered unacceptable based on steric problems in the final models. The second
strategy resulted in multiple solutions, which included, for most
models, the residues structurally equivalent to the amino acids known
to be critical for SEB binding to MHCII. The highest scoring solution
from the second strategy was chosen for further optimization. The
side-chain conformations for all residues were adjusted within the
Biopolymer module, and the final models were energy-minimized with 20 steps of Simplex and 100 steps of a gradient (Pullman's method), using
the Tripos force field (32).
To test the docking strategy, we built models of Spe-A/DR1, SEA/DR1,
and SEC3/DR1 complexes. Analysis of the possible solutions revealed
high scoring models, as judged by total energy and minimal steric
problems, with potential binding residues that were structurally equivalent to SEB amino acids critical for MHCII binding. Predictions from the SEA/DR1 complex model were verified by previously published experimental data (16). Based on these test results, the strategy was
assumed to be valid for searching for possible binding modes of Spe-C
to DR1.
 |
RESULTS |
The Spe-C superantigen was cloned from a clinical isolate of
S. pyogenes. r wt and mutants thought to be important
for binding to MHCII molecules (Fig. 1)
(discussed below) were also constructed. To avoid dimerization because
of covalent, intermolecular disulfide formation, all proteins had the
mutation C27S introduced. The C27S mutation did not affect Spe-C
biological function (discussed later) and allowed us to accurately
measure the chemical stability and propensity to form a non-covalent
dimer. Recombinant proteins were expressed in E. coli as
inclusion bodies and refolded on-column under non-reducing conditions.
The proteins were homogeneous as judged by SDS-PAGE (data not shown).
Similar results were obtained when the mutants were analyzed by LC-MS,
plotting the A280 and total ion current as a
function of retention time (data not shown). The summary of mass
assignments from LC-MS analysis is given in Table
I. The experimental and calculated masses
differ by less than four mass units, which excludes modifications due
to post-translational modifications by E. coli and
purification artifacts due to the refolding or chromatography
procedures.

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Fig. 1.
Residues of Spe-C mutagenized in the current
work. The zinc-binding site residues (black,
bottom left) and the putative MHCII -chain-binding site
residues (black, top right) overlaid on the -C
backbone (red) of Spe-C are shown. The figure was generated
with Swiss PDB Viewer v.3.7 (b2) (26).
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In view of reports indicating a possible dimer formation by the wt
Spe-C, the recombinant proteins were analyzed by size-exclusion chromatography. A typical profile is shown in Fig.
2, and the experimental molecular masses
of the main peaks are summarized in Table
II. Assuming dissociation constant,
Kd, of 0.390 mM for the dimer (3), the
expected amount of monomer at 0.2 mg/ml (8.13 µM) protein
concentration used in our size-exclusion chromatography experiments was
about 97%. The slightly lower (93%; see Table II) proportion of the
monomer may have been because of trimer formation, which was not
included in our calculations. These results indicate that only a small
population of dimer was present under our experimental conditions.

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Fig. 2.
Gel filtration chromatography of recombinant
H201A mutant at pH 7.0. Traces for other proteins were similar and
were excluded for clarity. The small peak at ~14.5 ml
corresponds to dimer, and the peak at ~19.5 ml corresponds
to the solvent. Protein concentration was 0.2 mg/ml.
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We next measured the far-UV circular dichroism spectra to characterize
the quality of in vitro refolded recombinant proteins. The
spectrum of r wt protein (Fig. 3)
exhibits a minimum at 210 nm and a second, less pronounced minimum at
about 220 nm. These data suggest a combination of
-helix and
-sheet as observed in the x-ray structure of Spe-C (2, 3). Estimates
of secondary structure were as follows: 31% for
-helix, 21% for
-sheet, and 48% for coil. These experimental values, within the
experimental error range, agree with the x-ray data (2, 3) as follows: 18 and 46% for the
-helix and
-sheet, respectively. The overall shape of the plots and absolute values of molar elliptically for the r
wt protein are also very similar to the spectrum of the closely related
Spe-A superantigen purified under non-denaturing conditions (14).

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Fig. 3.
Far-UV circular dichroism spectra of
recombinant wild type and mutant proteins. Solid line,
recombinant wild type; short dots, H201A; long
dots, H167A; long dash, D203A; short dash,
D203N. Protein solutions were prepared in 50 mM potassium
phosphate, pH 7.0, at a concentration of 0.2 mg/ml.
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Based on the far-UV CD spectra of Spe-C mutant proteins, the
secondary structure appeared to be similar to the r wt protein. However, the results for the H201A mutant may indicate potential folding problems in this protein. To further investigate, we measured the chemical stability of all recombinant proteins by GdnHCl unfolding. All proteins exhibited a cooperative transition under neutral pH
conditions indicating a well folded species. The midpoint of transition, Cm, was 1.10-1.16 M for
most proteins (Table III) and 0.92 M for the H201A mutant. The free energy of unfolding,
Go, was 30-31 kJ/mol for the r wt and H167A
mutant. The
Go of the other mutants, however,
were 24-26 kJ/mol. The differences, compared with r wt, were small but
reproducible and may indicate that some of the zinc binding residues
are important for protein stability.
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Table III
Thermodynamic parameters for equilibrium unfolding of Spe-C mutant
proteins
Protein concentration was 0.817 µM in 50 mM
potassium phosphate; pH 7.0. Thermodynamic parameter values are
average ± S.E. of two-four independent measurements.
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Spe-C binding to soluble MHCII was measured by surface plasmon
resonance, using HLA-DR1 expressed in S2 insect cells (24) and
complexed with the influenza hemagglutinin peptide 306-318 (25). The r
wt Spe-C bound HLA-DR1 with a dissociation constant of 1.0 µM whereas binding of zinc-binding site mutants was below the limits of detection (Table IV).
Inclusion of EDTA in the analyte greatly inhibited binding (Fig.
4), suggesting zinc dependence. The
residual binding (Fig. 4, inset) may indicate a secondary binding site on Spe-C for HLA-DR1. However, the signal was too weak to
estimate the Kd reliably. We next measured the affinity of recombinant Spe-C mutants for MHCII molecules expressed on
the cell surface to determine whether zinc was necessary for binding to
the expressed receptors. All the Ala-substituted mutants of Spe-C had a
significantly reduced affinity for the MHCII molecules expressed on LG2
cells (Fig. 5). The largest reduction was
observed for the D203A mutant. Surprisingly, the affinity of the D203N mutant was five times greater than that of the r wt
(Kd ~0.5 µM). The negative charge at
position 203 was apparently not necessary for the MHCII binding.

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Fig. 4.
Chelation of divalent cations by EDTA
dramatically reduces binding of HLA-DR1 to immobilized recombinant wild
type Spe-C as measured by surface plasmon resonance. Main
figure, an overlay of all sensograms. Inset, a close-up
of the binding sensograms in the presence of increasing concentration
of EDTA (37 °C, 10 mM HEPES, pH 7.0, 150 mM
NaCl). The HLA-DR1 concentration was 2 µM in all
experiments to improve signal to noise ratio for sensograms in the
presence of EDTA. For clarity, the data corresponding to start and end
of the injection were removed from the graph. The x axis
corresponds to time (s).
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Fig. 5.
Competitive binding of recombinant Spe-C
proteins to human LG2 cells. , recombinant wild type Spe-C;
, H167A; , H201A; , D203A; , D203N. Cells were incubated
with a mixture of recombinant wild type Spe-C labeled with FITC and
non-labeled Spe-C proteins. Receptor-bound fluorescence was measured by
flow cytometry.
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To exclude the potential contribution to our binding data of
HLA-DQ molecules co-expressed on the surface of LG2 cells, we performed binding studies with mouse L cells transfected with HLA-DR1
molecules. A representative result is shown in Fig.
6. Binding of the labeled r wt protein to
the cell surface of HLA-DR1 transfected cells was blocked by adding
increasing amounts of unlabeled Spe-C. The apparent
Ki, determined as the concentration value at 50% of
relative binding saturation, was 0.15 µM, a value lower
than the Ki of 0.5 µM estimated from
binding to human LG2 cells. The lower value may be because of the
contribution of different sets of peptides bound by HLA-DR1 expressed
on independent cell backgrounds. These results confirm that the binding
data reflected direct interactions between HLA-DR1 and Spe-C. We
concluded that the residues His167, His201, and
Asp203 are important for binding to MHCII molecules
expressed on cells and in solution. Additionally, metal, most likely
zinc, was essential for the overall binding of Spe-C to HLA-DR1, based
on our metal content analysis (Table V)
and cellular and surface plasmon resonance binding data. However, any
potential secondary, zinc-independent MHCII-binding site was likely
masked by the higher affinity, zinc-dependent site.

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Fig. 6.
Wild type Spe-C binds exclusively to
recombinant HLA-DR1 molecules expressed on the surface of cells.
Cells were incubated with a mixture of FITC-labeled recombinant wild
type Spe-C and non-labeled competitor proteins. Receptor-bound
fluorescence was measured by flow cytometry. , mouse L cells
transfected with HLA-DR1; , non-transfected mouse L cells.
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Table V
Zinc content of Spe-C proteins
Zinc content values are an average ± S.E. of at least two
independent measurements. Nickel content was below the detection limit
of the instrument. Under the experimental conditions, the minimum
detectable metal content was 0.01-0.03 mole of metal/mole of protein.
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To investigate whether replacements in the zinc-binding site of Spe-C
would affect the biological activity of the protein, we next measured
human T-cell stimulation. The r wt protein was first compared with wild
type SEA and Spe-A. SEA binds to the
-chain of MHCII molecules in a
zinc-dependent mode and to the
-chain in a
zinc-independent mode (15, 16), whereas the zinc-binding Spe-A has been
proposed to bind only to the
-chain (17). A typical T-cell
stimulation assay for the wild type proteins is shown in Fig.
7A. The r wt superantigen
produced by E. coli exhibits a typical superantigen response
curve with P50% (P50% is defined as the
concentration of antigen at which the T-cell stimulation is equal to
half of the maximum T-cell response) between 10 and 100 ng/ml. The
concentration of r wt toxin at which the response becomes saturated is
also slightly higher than observed for the native SEA and Spe-A. To
assess the importance of zinc-binding site for the T-cell stimulation
potential of Spe-C, we next examined mutants of the zinc binding
residues (Fig. 7B). The response point (the response point
is defined by us as the lowest concentration of antigen at which the
T-cell response is significantly higher than the baseline response) and
P50% values of the H167A mutant were identical to the wild
type protein. The H201A mutant had the same response point, but the
P50% value was slightly higher. However, the response
points of D203A and D203N mutants were higher than r wt values by an
order of magnitude. The saturation values were equal or slightly higher
than observed for the r wt protein. Clearly, none of the zinc-binding
site mutations except at position 203 caused a dramatic decrease in
biological activity. The replacement of Asp203 by either
Ala or Asn caused a noticeable decrease in initial T-cell recognition.
The T-cell response to the other mutants was essentially the same as
obtained with the r wt protein. The difference between T-cell
stimulation and MHCII binding by Spe-C may be a consequence of
stabilizing interactions with cell surface proteins other than MHCII
molecules (11). However, a major contribution by other proteins is
unlikely in view of the experiments with the HLA-DR1 transfectants
(Fig. 6) showing an exclusive binding to HLA-DR1. Although the
Asp203 mutations resulted in only a slight change in
protein-folding stability (Table III), we could not exclude the
possibility that mutating this residue may perturb local secondary
structure more than mutations in the other zinc-binding side chains.
Collectively, these data suggest that zinc binding is not essential for
T-cell recognition by Spe-C.

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Fig. 7.
Human T-cell stimulation by Spe-C is not
dependent on zinc. A, wild type superantigens are shown
as follows: , wild type Spe-C; , wild type SEA; , wild type
Spe-A. B, recombinant Spe-C proteins are shown as follows:
, wild type; , H167A; , H201A; , D203A; , D203N.
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It is conceivable that in addition to the high affinity zinc-binding
site observed in the crystal structure of Spe-C and MHCII complex (3),
there may be a second, lower-affinity binding mode important for T-cell
stimulation. We used protein docking simulations to facilitate a search
for additional MHCII-binding sites in Spe-C that were
zinc-independent. Several clusters of candidate binding sites were
identified, generally close to the hydrophobic loop (discussed later).
The final model structure, chosen based on the least amount of steric
problems, was the solution most favored by the computational search.
This solution also had the best agreement with our experimental data.
The orientation of Spe-C versus DR1 in the modeled complex
is very similar to the SEB/DR1 complex (root mean square deviation
between the complexes is 0.53 Å for the
-C atoms). There are two
major contact sites in the Spe-C/DR1 model complex: a hydrophobic loop
(Fig. 8A), containing no
intermolecular hydrogen bonds, and a polar pocket (Fig. 8B).
Both sites form an almost continuous binding surface. The side chain of
residue Thr34 from the hydrophobic loop packs very tightly
against the peptide backbone of Ser19-Gln18
from the
-chain of DR1. The polar pocket residue Tyr76
is on a flexible loop. This loop also contacts the following residues
from the TCR V
2.1 chain (13): Ile77 from the
complementarity-determining region 2/frame-work region 3 (CDR2/FR3),
Leu78 from CDR1, CDR2/FR3, and hypervariable 4/FR3 regions,
and Asn79 from CDR1 and CDR3 regions of TCR V
2.1. An
overlay of our modeled DR1/Spe-C complex on the published Spe-C/TCR
V
2.1 complex crystal structure (13) revealed no steric clashes
between HLA-DR1 and TCR V
, suggesting the likelihood of a ternary
complex of Spe-C/MHCII/TCR based on this alternative binding mode. It
should be noted that the zinc-binding site and hydrophobic loop/polar
pocket regions proposed to contact HLA-DR1 are on opposite surfaces of
Spe-C (Fig. 1).

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Fig. 8.
Close-ups of the hydrophobic loop
(A) and polar pocket (B) regions in
Spe-C proposed to bind HLA-DR1 -chain.
The backbone atoms of DR1 and Spe-C are colored in blue and
yellow, respectively. For clarity, only selected residues
are shown.
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We next examined two sets of mutants that targeted the sites shown to
be important for binding of Spe-C to the MHCII
-chain, based on our
modeling results. The first set, Y76A and Y87A, were mutations of
residues in the polar pocket, and the second, S32A, T33A, T34A, and
H35A, consisted of the hydrophobic loop mutations. Changes in position
76 of Spe-C were predicted to affect T-cell stimulation by influencing
the positioning of Spe-C in the ternary superantigen/MHCII/TCR complex.
The Spe-C side chain of His35, homologous to
Phe47 of SEB, in the Spe-C/DR1 model is within 4 Å of the
Ala37 and Met36 side chains of DR1
-chain.
The interaction is dominated by hydrophobic contacts with the side
chain of Met36 and the Met36-Ala37
peptide backbone. Mutation of these hydrophobic residues was anticipated to diminish binding to MHCII. The SEB loop residues Phe44 and Tyr46 do not have structural
equivalents in Spe-C. Overall, Ala replacements in the putative
-chain-binding site in Spe-C had minimal effects on protein
secondary structure (data not shown) and thermodynamic stability (Table
VI). The Y87A protein had slightly lower
thermodynamic stability. The residue 87 in Spe-C is very conserved in
the protein structure of other bacterial superantigens (discussed
later) and may form a hydrogen bond with the side chain of
Ser182, stabilizing an
-helix/
-sheet interaction.
Replacing the Ser182 side chain was expected to have a
negative effect on the overall stability of the protein. The
replacement Y76A or Y87A had a small effect on detectable binding
(Table VII). The T-cell stimulation, however, was diminished by the Y76A mutation, whereas the Y87A mutation
had no effect (Fig. 9A). The
effects of replacements in the hydrophobic loop on MHCII binding were
dependent on the position (Table VII). Calculated
Kds were higher for all mutants compared with the r
wt Spe-C, with the greatest effect on affinity noted for T33A and T34A.
T-cell stimulation by all loop mutants was also reduced (Fig.
9B), the most for T34A and H35A, and slightly less for the
T33A and S32A mutants. Our results clearly demonstrate that the
hydrophobic loop and the polar pocket in Spe-C are important for T-cell
stimulation.
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Table VI
Thermodynamic parameters for equilibrium unfolding of Spe-C mutant
proteins
Protein concentration was 0.817 µM in 50 mM
potassium phosphate, pH 7.0. Thermodynamic parameter values are
average ± S.E. of two-five independent measurements.
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Table VII
A summary of affinities of recombinant HLA-DR1 for Spe-C protein
variants as measured by surface plasmon resonance
The rate constants were determined at 37 °C in 10 mM
HEPES, pH 7.0, 150 mM NaCl. The equilibrium dissociation
constant for wt, determined from the plot of apex of the on-rates
versus DR1 concentration, was between 1 and 2 µM. The
value did not change when 10 mM potassium phosphate, pH
7.0, was used instead of the HEPES buffer. The data for wt protein was
included for comparison.
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Fig. 9.
Alternative zinc-independent binding surface
of Spe-C controls T-cell recognition. A, stimulation of
human T-cells by recombinant polar pocket mutants. , recombinant
wild type; , Y87A; , Y76A. B, stimulation of human
T-cells by hydrophobic loop mutants of Spe-C. , recombinant wild
type; , S32A; , T33A; , T34A; , H35A.
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|
 |
DISCUSSION |
Bacterial superantigens bind to MHCII receptors through one or
both of two conserved sites and also to TCR molecules through more
variable sites (1). Dimerization of Spe-C was proposed previously (2)
to bury the potential MHCII
-chain-binding site in the subunit
interface, resulting in interaction with only the MHCII
-chain, a
mechanism for binding to MHCII that differs considerably from all other
superantigens. In the present study, mutation of the Cys27
to Ser eliminated potential covalent dimerization, without altering biological activity. Although we detected only a low proportion of
dimer, superantigen interaction with surface-bound MHCII molecules may
be conducive to aggregate formation. Clarification of the biologically
active unit on the cell surface awaits further study. Nonetheless, our
results suggest that the zinc-mediated interactions between Spe-C and
MHCII observed in the x-ray structure of a co-complex (3) may reflect
conditions conducive for crystallization but not necessarily the mode
of interaction required for TCR signal transduction. In our study,
binding of Spe-C to MHCII was greatly diminished when the zinc binding
residues of Spe-C were replaced by alanines. Although these mutations
caused a minor change in
Go, the Spe-C
mutants retained T-cell stimulatory activity. In a previous report
(18), Ala replacements of zinc binding residues in SEE/SED caused a
significant decrease in thermal stability of the mutant proteins and
abolished T-cell recognition, indicating an important role for
zinc in stabilizing protein folding of some superantigens.
To search for a potential zinc-independent binding mode for Spe-C, we
first analyzed the structural features of superantigen protein surfaces
known or suggested to be involved in MHCII and TCR binding. The
zinc-binding site is structurally conserved in many superantigens, as
is the overall superantigen fold. Based on the available x-ray data, we
constructed homology models of other zinc-binding superantigens for
which the experimental data for the structure of these proteins was not
available. A structural alignment extracted from these superimposed
models is presented in Fig. 10.
Mutation of the hydrophobic loop region in several superantigens
inhibits binding to MHCII molecules
(15).2 For superantigens that
are hypothesized to use an MHCII binding mechanism differing
from SEB, i.e. are not known to insert into the hydrophobic
binding surface of DR1, either the loop has a different conformation
than in SEB, for example Spe-C, or it is occupied by
small/non-hydrophobic residues, for example those found in Spe-G,
Spe-J, Spe-H, and SMEZ-2 (Fig. 10). The zinc-binding site and polar
pocket residues appear to be conserved when the hydrophobic binding
loop is defective. The loop of Spe-C, alone or in conjunction with
other structurally conserved residues, may be involved in a low
affinity binding to MHCII molecules. However, because the structural
composition of the loop is not optimal for the
-chain binding in the
mode observed in SEB/MHCII complex (19), either the surface of the
superantigen/MHCII interface differs, as suggested for the SSA/HLA-DQ
complex (20), or the relative orientation of the MHCII and superantigen
molecules differs from the SEB/MHCII complex, as observed in TSST-1/DR1
complex (21). The hydrophobic loop residue His35 and polar
pocket residues Tyr76 and Tyr87 of Spe-C are
very conserved in all S. pyogenes superantigens (Fig. 10).
The His35 residue in Spe-C may contribute to the polar
pocket through its proximity to the residue Glu54 (Fig.
8A). In addition, the lower free energy of unfolding for the
H35A mutation (Table V) indicates that His35 stabilizes the
loop structure. The residues structurally equivalent to
Thr34 of Spe-C in other S. pyogenes
superantigens (Fig. 10) are Thr (Spe-C, Spe-G, Spe-J) or Ser (Spe-I,
Spe-H, Smez-2, Spe-A), possessing both hydrophobic and polar qualities.
The Thr33 residue is involved in stabilizing the loop
through intramolecular H-bonds, hence the T33A change may destabilize
the loop. The replacement S32A may affect the conformation of the loop
by removing a potential H-bond interaction with carbonyl oxygen from
the Glu31-Ser32 peptide bond. Both changes have
a negative effect on the productive binding to DR1 leading to T-cell
activation as observed in our current work (Fig. 9B).

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Fig. 10.
Structural alignment of known and predicted
bacterial superantigen sequences in the vicinity of the MHCII
-chain binding residues of SEB protein.
Top, the hydrophobic loop region. Bottom, the
polar pocket region. For clarity, only the residues structurally
equivalent to Spe-C Tyr76 and Tyr87 are colored
in magenta. The -C backbones of structures were overlaid
with Swiss PDB Viewer v.3.7 (b2) (26) program, and residues within 4 Å root mean square deviation of the corresponding amino acids of
-chain binding loop of SEB are colored in magenta and
underlined (top) or colored in magenta
only (bottom). Numbers on the top
of alignments refer to residues in Spe-C protein. The sequence names
corresponding to S. pyogenes superantigens are marked in
red. The structures were either modeled by Swiss Model as
described under "Materials and Methods" or downloaded from a PDB
data base (marked with an asterisk by the name). The
proteins and the corresponding PDB codes are as follows: SMEZ-2, 1EU3;
SEB, 1SEB; Spe-H, 1EU4; SSA, 1BXT; TSST-1, 2QIL; SEA, 1SXT; SEH, 1HXY;
SEC3, 1JCK; and Spe-A1, 1B1Z. **, structure of SED was solved (33), but
the coordinates were not publicly available. Therefore, a model of SED
was built as described under "Materials and Methods." Sequences for
the modeled S. pyogenes proteins were derived from the
Genome Sequence Data base (accession number AE004092) as
described by Ferretti et al. (34). Accession codes for other
modeled proteins were as follows: SED (AAB06195), SEG
(BAB42910), SEI (AAC26661), SEJ (AAC78590), S.E. (AAG36952), SEN
(AAG36956), SEO (AAG36951), and SEP (BAB43036).
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In the polar pocket of Spe-C, the Tyr76 and
Tyr87, together with Glu54, are proposed to
bind to Lys39 of
-chain of DR1 (Fig. 8B). The
residues are highly conserved in other superantigens (Fig. 10), and
structurally equivalent residues are either observed to form a H-bond
with Lys39 of
-chain of DR1 in SEB/MHCII (19) complex or
postulated to form such a bond in the SEA/MHCII (16) complex model. The
Y76A replacement decreased T-cell stimulation (Fig. 9A)
whereas the mutation Y87A had no effect. In SEA, the mutation Y108A,
structurally equivalent to the Y87A mutation in Spe-C, also had no
effect on HLA-DR1 binding (16). However, the Y92A mutation in SEA,
structurally equivalent to the Y76A mutation in Spe-C, decreased both
DR1 binding and T-cell stimulation (16). The striking similarity in
results obtained with both superantigens suggests that MHCII binding by the Y76A mutant of Spe-C was also diminished but masked by the dominant
zinc-dependent interactions with MHCII
-chain. An
alternative explanation is that the mutation Y76A, but not Y87A, has
altered TCR interactions with the Spe-C/MHCII complex. However, TCR
contacts with this surface of Spe-C also precludes the active role of a Spe-C homodimer as observed by Li et al. (3). Last,
stability of the homodimer may have been affected by the mutations
studied, yet dimer stability is not likely to have an influence T-cell stimulation, as the reported Kds for dimer formation by wt and H35A mutant proteins (3) are several orders of magnitude above the physiologically relevant Spe-C concentration.
In summary, our experimental results clearly demonstrate a diminished
role of zinc and dimerization in the biological activity of Spe-C. The
high affinity, zinc-binding site may be important for Spe-C binding to
MHCII molecules but not directly for T-cell stimulation. The
zinc-dependent affinity may serve as a mechanism to
increase the local superantigen concentration (16), mimicking the
effect achieved by receptor oligomerization (23). A second site is
proposed to be involved directly in T-cell stimulation by positioning
Spe-C on the MHCII surface for biologically relevant activity,
facilitating superantigen alignment in the Spe-C/MHCII/TCR ternary complex.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Senior fellow of the National Research Council. To whom
correspondence may be addressed. Tel.: 301-619-4811; Fax: 301-619-2348; E-mail: wes.swietnicki@amedd.army.mil.
§
To whom correspondence may be addressed. Tel.: 301-619-4232; Fax:
301-619-2348; E-mail: ulrich@ncifcrf.gov.
Published, JBC Papers in Press, December 8, 2002, DOI 10.1074/jbc.M206957200
2
R. G. Ulrich, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
Spe, streptococcal pyrogenic exotoxin;
CDR, complementarity-determining
region;
FITC, fluorescein isothiocyanate;
GdnHCl, guanidine
hydrochloride;
HLA, human leukocyte antigen;
LB, Luria-Bertoni broth;
LC-MS, liquid chromatography-mass spectrometry;
MHCII, major
histocompatibility complex type II;
r wt, recombinant C27S mutant of
Spe-C protein;
SE, staphylococcal enterotoxin;
TCR, T-cell receptor;
TSST, toxic shock syndrome toxin;
wt, wild type.
 |
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