Crystallographic and mutational data show that the streptococcal pyrogenic

exotoxin J (SPE-J) can use a common binding surface for T-cell receptor

binding and dimerization.

Heather M. Baker1,2, Thomas Proft2,3, Phillip D. Webb3, Vickery L. Arcus1,2, John D.

Fraser2,3 and Edward N. Baker1, 2*.

1School of Biological Sciences and 2Centre of Molecular Biodiscovery, University of

Auckland, Private Bag 92019, Auckland, New Zealand.

3Department of Molecular Medicine, University of Auckland, Private Bag 92019, Auckland,

New Zealand.

*Corresponding author

Phone: +64-9-373-7599

Fax: +64-9-373-7619

Email: ted.baker@auckland.ac.nz

JBC Papers in Press. Published on July 7, 2004 as Manuscript M406695200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Running title: Structure and function of the superantigen SPE-J

Key words: Superantigen, T-cell receptor binding, mutagenesis, dimerization, crystal

structure.

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SUMMARY

The protein toxins known as superantigens (SAgs), which are expressed primarily by the

pathogenic bacteria Staphylococcus aureus and Streptococcus pyogenes, are highly potent

immunotoxins with the ability to cause serious human disease. These SAgs share a conserved

fold, but quite varied activities. In addition to their common role of crosslinking T-cell

receptors (TCRs) and MHC class II molecules (MHC-II), some SAgs can crosslink MHC-II,

using diverse mechanisms. The crystal structure of the streptococcal superantigen SPE-J has

been solved at 1.75 Å resolution (R = 0.209, Rfree = 0.240), both with and without bound

Zn2+. The structure displays the canonical 2-domain SAg fold and a Zn-binding site that is

shared by a subset of other SAgs. Importantly, in concentrated solution and in the crystal,

SPE-J forms dimers. These dimers, which are present in two different crystal environments,

form via the same face that is used for TCR binding in other SAgs. Site-directed mutagenesis

shows that this face is also used for TCR binding by SPE-J. We infer that SPE-J crosslinks

TCR and MHC-II as a monomer, but that dimers may form on the antigen-presenting cell

surface, crosslinking MHC-II and eliciting intracellular signalling.

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INTRODUCTION

The common human pathogens Staphylococcus aureus and Streptococcus pyogenes secrete a

number of potent protein toxins known as superantigens. These toxins derive their name from

their primary functional attribute, which is to bind simultaneously to T-cell receptors (TCRs)

and MHC class II (MHC-II) molecules, outside the MHC peptide binding groove and as

intact molecules rather than processed peptides. This can cause massive overstimulation of the

cellular immune response, with the overproduction of cytokines such as tumor necrosis factor

α and interleukin-2, as a result of uncontrolled T-cell activation (1-3). This activity is central

to their involvement in many human diseases, such as toxic shock, scarlet fever, food

poisoning, and possibly others such as rheumatoid arthritis (2, 4, 5).

The SAg family comprises staphylococcal enterotoxins (SEs) such as SEA, SEB, SEC1-3

and SED, streptococcal pyrogenic exotoxins (SPEs) such as SPE-A and SPE-C, and toxic

shock syndrome toxin-1 (TSST-1). The sequencing of the complete genomes of several

strains of S. aureus (6) and S. pyogenes (7) has led to the discovery of many more sag genes,

including spe-j, and the realisation that in these two organisms this is a widespread protein

family that must play a major role in their pathogenicity. The SAgs share widely different

levels of sequence identity. Some are so similar (for example SEA and SEE, with ~ 90%

sequence identity) as to make allelic variants between different strains difficult to distinguish,

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but many share much lower sequence identity, around 20%. Structurally, however, the SAgs

share a highly conserved fold (8, 9), comprising an N-terminal β-barrel domain with the

well-known OB-fold (10, 11), and a C-terminal β-grasp domain comprising a β-sheet that

wraps around a long central helix.

A striking feature of the SAg family, however, is that this conserved fold supports a wide

variety of different binding modes. Most SAgs (for example SEB and TSST-1) have a single

MHC-II binding site, located on their N-terminal domains (12-14), often referred to as the

generic MHC-II binding site, whereas others (such as SMEZ and SPE-H) have instead a site

on their C-terminal domains, mediated by a bound Zn2+ ion (9). Still others, such as SEA,

have both sites (15-17), giving them the ability to crosslink MHC-II on antigen-presenting

cells (APCs) and thus elicit intracellular signalling in the APCs. A variation on this theme is

given by several other SAgs, including SED and SpeC, which can crosslink MHC-II by

formation of homodimers. Thus, SED forms Zn-dependent homodimers through its C-

terminal domain, and can crosslink MHC-II through the N-terminal domain sites at each end

of the homodimer (18). On the other hand, SPE-C dimerizes via its N-terminal domain, and

can crosslink MHC-II by the two C-terminal domain Zn2+ sites of the dimer (19).

In contrast to the varied MHC-II binding modes, the evidence so far suggests that most, if not

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all, SAgs bind to TCR via a common site, at the interface between the N- and C-terminal

domains (20, 21). The ability to select particular TCR Vβ sub-types appears to derive from

sequence and structural diversity on the TCR, coupled with local SAg sequence variation at a

common TCR binding site.

Streptococcal pyrogenic exotoxin J (SPE-J) was first identified from the S. pyogenes genome

sequence (22). The recombinant protein has been shown to be a highly potent mitogen, giving

half maximum responses at 0.1 pg/mL. In terms of sequence, SPE-J is most closely related to

SPE-C (49% identity) and it appears to be functionally indistinguishable; its T-cell

specificity is the same (for Vβ2.1) and like SPE-C (23) it forms homodimers and induces

rapid homotypic aggregation of LG-2 cells, implying an ability to crosslink MHC-II (24).

This raises the question as to why a bacterial isolate should maintain two different genes that

code for apparently functionally identical proteins.

Here we show, from its high resolution crystal structure, that SPE-J forms a completely

different dimer from that of SPE-C. Intriguingly, the interface used for dimerization proves to

be the same as that used for TCR binding. This leads to the conclusion that SPE-J must bind

to TCR as a monomer, but that concentration-dependent dimerization allows it also to

stimulate intracellular signalling in APCs by crosslinking MHC-II as a dimer.

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EXPERIMENTAL PROCEDURES

Protein Expression and Purification

SPE-J was cloned and expressed in E.coli as described by Proft et al. (24). The protein was

overexpressed as a glutathione S-transferase (GST) fusion protein, and was initially purified

using glutathione/agarose. After cleavage of GST from the toxin with protease 3c, the protein

was further purified by cation exchange chromatography (MonoS HR 5/5 column, Pharmacia)

followed by gel filtration (Superdex 75 HR 10/30 column, Pharmacia). Small fractions were

taken across the protein peak and dynamic light scattering (see below) was used to decide

which fractions were to be taken for crystallization trials. Only those with a Cp/Rh ratio of

less than 14% were used.

Light scattering analysis.

Dynamic light scattering was performed using a Protein Solutions (Charlottesville, VA)

DynaPro molecular sizing instrument to determine not only the monodispersity of protein

samples, prior to crystallization, but also to determine the relative molecular mass of the

protein at various concentrations. Samples ranged in concentration from 0.8 to 12.0 mg/mL

and 30 measurements were made at each concentration. Results are summarized in Table 1.

Crystallization

Crystals were grown at 18ºC by the hanging drop method by mixing 1 µL of protein solution

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(10 mg/mL protein in 50 mM HEPES/KOH, 100 mM NaCl, pH 7.0) with 1 µL of reservoir

solution (210 mM lithium acetate, 17% PEG 3350, pH 5.5). Small shield-shaped crystals of

maximum dimension 0.03 mm grew over a period of two to three weeks. These crystals were

monoclinic, space group C2, with unit cell dimensions a = 165.6, b = 46.4, c = 72.2 Å, ß =

90.6º. This gave VM values of 3.0Å3/Da (59% solvent) assuming two molecules per

asymmetric unit, or 2.0 Å3/Da (39% solvent) assuming three molecules per asymmetric unit;

the structure determination showed the latter to be correct.

Crystals of Zn-bound SPE-J (Zn-SPE-J) were obtained by soaking crystals in 100 mM zinc

acetate, 20% PEG 3350, 230 mM lithium acetate, pH 5.8, for one hour. This short, sharp soak

gave much better diffraction than that from crystals soaked in lower zinc acetate concentration

(1 mM) for a longer period (4 - 24 hours). Crystals were mounted in a cryoloop and flash-

frozen by plunging into liquid N2 after a rapid pass through a cryoprotectant solution. The

latter comprised 0.23 M lithium acetate, 18% PEG 3350 and 20% ethylene glycol, pH 5.8, for

SPE-J, and 100 mM zinc acetate, 0.23 M lithium acetate, 20% PEG 3350 and 20% ethylene

glycol, pH 5.8, for Zn-SPE-J.

Data Collection.

X-ray diffraction data to 1.7 Å resolution were collected for SPE-J at 110K at the SSRL

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(Stanford Synchrotron Radiation Laboratory). Zn-SPE-J data to 2.0 Å resolution were

collected at 110K using CuK± radiation from a Rigaku RU-H3R X-ray generator equipped

with Osmic mirrors, an Oxford cryostream and a Mar345 imaging plate system. Raw data

were processed using MOSFLM (25) and scaled and merged with SCALA (26). Data

collection statistics are summarized in Table 2.

Structure Determination and Refinement

The structure of SPE-J was solved by molecular replacement using AMoRe (27) with the

closely related SAg structure SPE-C (19) (PDB code 1AN8), as search model; SPE-J and

SPE-C share 49% sequence identity. Two molecules were found and used for phasing to 2.0

Å resolution, after which an initial model was built with ARP/wARP (28). This gave an

almost-complete model for both molecules (391 out of 422 residues) and also revealed the

position of a third molecule, which was added to the model. Further refinement was with CNS

(29), with cycles of refinement being interspersed with manual model building into the

electron density using the graphics program TURBO FRODO (30). Solvent molecules, all

treated as water, were added using the WATERPICK facility in CNS, and were retained if

they had spherical density and appropriate hydrogen bond geometry. The quality of the model

was checked periodically with PROCHECK (31) and hydrogen bonds identified following the

distance and angle criteria of Baker and Hubbard (32). The Zn-SPE-J structure was solved

using the final SPE-J structure as a starting model, and was refined in the same way.

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Site-directed Mutagenesis

Single-site mutants of SPE-J were generated by overlap PCR. Oligonucleotide primers

pGEX.fw/SpeJmut.rev and pGEX.rev/SpeJmut.fw were used for 12 cycles of PCR with

pGEX-3c:speJ (24) as template. See supplementary data for primer sequences. The PCR

products were then purified from agarose gels and used as templates for 18 cycles of PCR

with pGEX.fw/pGEX.rev primer pairs. The PCR products were cloned into pGEX-3c vectors

and the recombinant SPE-J mutant proteins were produced as previously described for

wildtype SPE-J (24). The DNA sequences of the cloned SPE-J mutants were confirmed

using a Licor automated DNA sequencer (model 4200).

Toxin Proliferation Assay

The mitogenic activity of the SPE-J mutants was determined in a peripheral blood

lymphocyte (PBL) stimulation assay as described previously (22). In brief, PBLs were

purified from blood of healthy donors and incubated with varying dilutions of SPE-J mutants

(100 ng/mL to 1 fg/mL). After 3 days of incubation at 37°C, 0.1 µCi [3H]thymidine was

added. After another 24 h, the PBLs were harvested and counted on a Cobra scintillation

counter. The decrease in T cell mitogenecity was calculated as the amount of mutant toxin

needed to achieve half maximum stimulation (P50 value) of wild type SPE-J.

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RESULTS

Crystal structure of SPE-J

The three-dimensional structure of SPE-J was determined by molecular replacement and

refined at 1.75 Å resolution to an R factor of 0.209 and free R factor of 0.240. The model has

excellent geometry with 87.2% of non-glycine residues falling in the most favored regions of

the Ramachandran plot, as defined in PROCHECK (31), with no outliers. The three molecules

in the asymmetric unit of the crystal are organised in such a way that A and B form a putative

dimer (see below) and C also forms a dimer, with another molecule C, related by two-fold

crystallographic symmetry. The final model for molecule A comprises the complete

polypeptide for mature SPE-J, residues 1-209, but with two additional residues (Gly-2 and

Ser-1) also modeled at the N-terminus, left after cleavage of the GST fusion domain.

Sequence numbering here follows that of the mature protein. Molecule B lacks residues 97-

101, and molecule C lacks residues -2, -1 and 1; these have no interpretable electron density

and are assumed to be disordered. Further details are in Table 2.

Molecular structure

SPE-J has the characteristic two-domain SAg fold (8, 9), shown in Figure 1. Following an

N-terminal helix α2 (residues 2-17), which ends in the inter-domain cleft, the N-terminal

OB-fold domain has a β-barrel structure comprising five highly curved β-strands, β1 to β5,

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which create a concave outer surface that is used in many SAgs for MHC-II binding. The C-

terminal β-grasp domain is based on a mixed, 5-stranded, β-sheet that wraps around a

central α-helix, α4 (residues 128-145). When SPE-J is compared with other SAgs using the

program SSM (http://www.ebi.ac.uk/msd-src/ssm/), its closest homolog is SPE-C, with

which it shares 49% sequence identity and with which 199 residues can be matched with a

root-mean-square (rms) difference in Cα positions of 1.24 Å. This gives a Z-score of 14.8,

with the only significant differences in the polypeptide chain conformation being in the β5-

β6 loop, where a single residue (Asn96) is inserted in SPE-J, and small changes in strand β7

and the β4-β5 and β10-α5 loops. The next closest hits are SMEZ-2 (Z-score 11.0, 32%

sequence identity, 196 Cα atoms matching with an rms difference of 1.41 Å) and SPE-H (Z-

score 9.0, 25% sequence identity, 182 Cα matching with an rms difference of 1.92 Å). These

are all streptococcal SAgs.

[Figure 1]

Molecular packing evidence for dimerization.

Examination of the crystal packing shows that molecules A and B share a significant interface

(Figure 2) which is considerably more extensive than any of their other packing interactions

in the crystal, and has many of the properties expected of a protein dimer. This interface

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buries a total of 1360 Å2 of accessible surface area (680 Å2 per monomer, or 6.5% of the

monomer surface), calculated using the Protein-Protein Interaction Server

(http://www.biochem.ucl.ac.uk/bsm/PP/server); this uses the algorithm of Lee and Richards

(33) with a probe radius of 1.4 Å. The interface is formed by the C-terminal half of helix α2

and its connection to strand β1, the β2-β3 loop, the β4-β5 loop, all from the N-terminal

domain, and the end of helix α4 and start of helix α5, both from the C-terminal domain. The

residues that make the greatest contribution to the interface are Tyr14, Glu17 and Ile19 from

α2, Phe77, Arg79 and Tyr83 from β4-β5, Gln142 from α4, and Arg181 from the start of α5,

which hydrogen bonds across the interface to the carbonyl oxygen of Gly17.

The third molecule in the asymmetric unit, molecule C, forms a very similar interaction with

another molecule C, related by crystallographic symmetry. The dimerization of these two

molecules buries a somewhat larger surface area of 2130 Å2 (1065 Å2 per monomer, 10.4%

of the monomer surface). The structural elements that make it up are the same as for the A-B

dimer, however, involving residues in and around the interdomain cleft (Figure 2). The

principal contributors to the interface are Tyr14, Glu17, and Ile19 from α2, Tyr43, Lys44, and

Lys45 from β2-β3, Phe77, Tyr80, and Tyr83 from β4-β5, Gln142 from α4 and Arg181 from

α5. In both the A-B and C-C dimers there are 6-10 direct protein-protein hydrogen bonds

across the interface, and a number of water molecules make bridging interactions.

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[Figure 2]

Zinc binding

SPE-J has been shown to bind to MHC-II in a zinc-dependent manner. The native SPE-J

structure contained no bound zinc, however, and of the three residues proposed to form the

Zn2+ binding site (24), the side chains of His201 and Asp203 were close together but that of the

third putative ligand, His167, was turned away. In the Zn-SPE-J structure, however, after

soaking the crystals very briefly in 100 mM Zn2+, the side chain of His167 had moved and

these three residues are bound to a fully-occupied Zn2+ ion, with bond lengths of 2.1 – 2.2 Å.

A water molecule is bound as a fourth ligand, completing a tetrahedral coordination site. The

zinc site is located on the concave surface of the C-terminal domain and is equally accessible

for MHC-II binding in both the monomeric and dimeric forms of SPE-J dimer. In the latter

the Zn atoms are ~ 60 Å apart, at the two ends of the dimer.

Functional analysis of the TCR binding site in SPE-J

SPE-J is most closely related to SPE-C by amino acid sequence (49% identity) and, like

SPE-C, primarily stimulates T-cells carrying the Vβ2 TCR (24). We therefore selected for

mutagenesis those residues in SPE-J that were equivalent to the SPE-C residues shown to

contact Vβ in the TCR Vβ-SPE-C co-crystal structure (21) (Table 3). The recombinant

SPE-J mutant proteins were then analyzed for their mitogenicity in standard PBL

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proliferation assays. To ensure that any loss in proliferation activity was due to impaired TCR

binding, MHC-II binding of the mutant proteins was confirmed in a standard binding assay

(data not shown).

The strongest decrease in potency for T-cell stimulation (10,000-fold) was observed with the

Y14A and R181Q mutants, which correspond to SPE-C residues Tyr15 and Arg181,

respectively. Ten-fold and 100-fold decreases in potency were detected with mutants K44A

(Arg45 in SPE-C) and F77A (Leu78 in SPE-C), respectively. A minor difference was

observed with T78A (Asn79 in SPE-C) which had a 2-fold reduction in mitogenicity

compared with wild type SPE-J. In contrast, the mutants E17A, I19A, K46A, F48A and

S178A showed no differences to wild type SPE-J in proliferation (Table 3).

DISCUSSION

Solution studies, using dynamic light scattering, show clearly that SPE-J forms dimers at

higher protein concentrations (> 3 mg/mL) and in fact has a somewhat greater propensity for

dimerization than SPE-C. In contrast, another SAg, SMEZ-2, showed only monomers under

the same conditions. SPE-J was also shown to stimulate the rapid aggregation of LG-2 cells,

presumably by crosslinking of MHC-II molecules (24). Further functional studies indicate

that SPE-J binds to MHC-II only through the C-terminal zinc site that it shares with other

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zinc-dependent SAgs (24), and the conclusion must be that, as in the case of SPE-C (23), it is

its ability to form dimers that enables SPE-J to crosslink MHC-II.

The mode of dimerization found in our SPE-J crystals was a surprise, however. It does not

involve the N-terminal OB-fold binding face, as is the case for dimers of SPE-C (19), nor

does it involve the face of the C-terminal β-sheet as for SED (18). Instead, the dimer

interface in SPE-J, as seen in the crystal structure, involves residues in and around the

interdomain region, residues 10-19 from helix α2 and the α1-β1 loop, 42-45 from the β2-

β3 loop, 77-83 from the β4-β5 loop, Gln138 and Gln142 from helix α4 in the C-terminal

domain and Arg181, from helix α5, also in the C-terminal domain. Although it cannot

necessarily be inferred that a mode of association seen in crystals also occurs in solution,

especially if the surface area buried by this association is not great (34), in the present case

SPE-J has been shown to form dimers in solution, at similar concentrations as were used to

grow the SPE-J crystals. Most significantly, the same dimer is found in two completely

different crystal environments, namely the A-B dimer between two independent molecules in

the asymmetric unit and the C-C dimer between two molecules related by crystallographic

symmetry. This argues strongly that the same dimer would be seen in solution. The buried

surface area (1360 Å2 for A-B and 2130 Å2 for the more closely-packed C-C dimer) is at

the low end of the range for functional dimers (34, 35), but is consistent with the solution data

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that shows dimerization only at higher concentrations.

The SPE-J mutagenesis results indicate that the TCR binding surface is very similar to that

for SPE-C, consistent with their high structural similarity. The strongest decrease in T-cell

mitogenicity was observed in the Y14A and R181Q mutants (10,000-fold). The equivalent

residues in SPE-C (Tyr15 and Arg181) hydrogen bond to the TCR β-chain (21) and in SEC3

mutation of the equivalent residues (Asn23 and Gln210) abrogates TCR binding completely

(36). Thus these residues appear to be key residues for TCR binding, and the strong

conservation between SPE-C and SPE-J may explain their shared T-cell specificity, as both

toxins primarily target the TCR Vβ2 chain. The mutational data also show that Phe77 makes

an important contribution to TCR binding by SPE-J, although like its equivalent in SPE-C

(Leu78) it can only make van der Waals contacts. The neighbouring residue in SPE-C

(Asn79) is hydrogen bonded to TCR Vβ, but mutation of Thr78 in SPE-J has little effect. We

conclude that the β4-β5 loop, to which these residues belong (as well as Tyr90, shown to be

important in SEC3 (36)), is important for TCR binding but through different residues in

different SAgs; indeed the loop conformation is slightly changed in SPE-J relative to SPE-C.

Most of the other residues mutated make only van derWaals interactions, and it is likely that a

single residue makes too small a contribution to the interface to significantly weaken binding

when truncated to alanine.

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What is most striking is that the surface that is used for binding to the T-cell receptor Vβ

chain during T-cell stimulation is essentially the same surface that is used for dimerization of

SPE-J (Figure 3). This surface, which is centered around the cleft between the N- and C-

terminal domains is the site of TCR Vβ binding for all the SAgs for which SAg-Vβ

complexes have been structurally characterised.

[Figure 3]

From a structural viewpoint, it is not unreasonable that the same (or very similar) surface can

be used to bind different molecules, as is shown in a recent analysis of protein-protein

interfaces (37). Such surfaces could be described as moonlighting surfaces, able to support

diverse protein-protein interactions, especially those that are transient or of relatively low

affinity. The interaction of SAgs with their TCR Vβ ligands is of this nature (KD ~ 10-4 to

10-6 M) (38) and the surface area buried is usually correspondingly small (1268 Å2 for

SEB-Vβ and 1324 Å2 for SPE-A-Vβ) (21). This is very similar to the surface area buried in

the SPE-J dimer, and this, too, is of low affinity as shown by its concentration dependence.

One of the fascinating features of proteins of the SAg family is their diversity of functional

behaviour. Among the true SAgs, with immunostimulatory activity, some have only a single

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MHC-II binding site, whereas others have two, and even when the same site is employed the

mode of MHC-II binding can vary significantly. Some SAgs can crosslink MHC-II, but by

different mechanisms. Some seem always to be monomeric, but others can dimerize, and in

different ways. Moreover in the wider SAg family there is a large group of toxins, the SETs,

that are clearly homologous, sharing the same fold and moderate sequence identity, but which

do not have superantigen activity (39). Instead, these proteins target other components of the

immune system (R. Langley, T. Proft and J. D. Fraser, unpublished). The SET family has

recently been renamed SSL (staphylococcal superantigen-like) to avoid confusion with the

prototypical SAgs (40). This diversity of activity in proteins with a common fold can arise

because of its dependence on relatively low affinity surface interactions, often employing the

same or similar binding sites, albeit with different detail.

What are the functional and physiological implications of the dimerization seen here for SPE-

J? The clear conclusion must be that SPE-J crosslinks MHC-II and TCR only as a monomer,

since the TCR-contacting residues are buried in the dimer. We also conclude, however, that

SPE-J can crosslink MHC-II molecules as a dimer, and so stimulate intracellular signalling

and cytokine expression by antigen-presenting cells, but independently of its TCR activation

ability. Which of these activities is expressed may depend on local concentration effects.

Seen in this light, the ability of SPE-J to express different activities under different

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conditions, stimulating T-cells as a monomer and crosslinking MHC-II as a dimer, is yet

another expression of the diverse behaviour of this family. It is reasonable to suppose that

other activities will be uncovered, even for apparently well-characterised family members. In

this connection, we note that in at least one crystal form of TSST-1 (41), a crystal dimer is

found that uses essentially the same binding surface as in the SPE-J dimer. The relative

orientations of the two TSST-1 molecules are different from those of the two SPE-J

molecules, but the interaction could imply that TSST-1, too, could express other activities

through this association, if replicated in solution. Again by analogy with SPE-J, the

interaction in the TSST-1 crystal dimer could provide a model for its TCR binding surface,

which has not yet been defined crystallographically.

ACKNOWLEDGEMENTS

This work was supported by the Health Research Council of New Zealand (grants to ENB and

JDF), the Royal Society of New Zealand Marsden Fund (Fast Start grant to TP), and the

Centre of Molecular Biodiscovery from the Centres of Research Excellence funding. We also

thank Dr. Clyde Smith and staff at the Stanford Synchrotron Radiation Laboratory for help

with data collection.

DEPOSITED DATA

The atomic coordinates and structure factors have been deposited in the Protein Data Bank

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(http://www.rcsb.org/) with accession codes xxxx (SPE-J) and yyyy (Zn-SPE-J).

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FIGURE LEGENDS

FIG. 1. Structure of the SpeJ monomer. Ribbon diagram in which the major secondary

structural elements are labeled in accord with the nomenclature first used for SEB (42). In this

orientation the N-terminal domain is on the right and the C-terminal domain on the left.

Secondary structural elements are: α2, 2-19; β1, 21-32; β2, 35-40; β3, 48-54; β4, 66-72;

β5a, 81-87; β5b, 88-92; β6, 102-108; β7, 111-118; β8, 125-127; α4, 128-145; β9, 155-162;

β10, 166-172; α5, 180-186; β11, 194-196; β12, 201-208. Figure drawn with Pymol (http://

FIG. 2. Dimerisation of SpeJ. Stereo view of the SPE-J dimer, with the two molecules shown

in blue and yellow. Side chains that make significant contributions to the dimer interface are

shown in magenta, with the major contributors labeled: Y Tyr14, I Ile19, F Phe77, and R

Arg181. The C-terminal domain in each monomer provides the binding site for the zinc ion

(grey sphere) which binds to MHC-II. Figure drawn with MOLSCRIPT (43), rendered with

RASTER3D (44).

FIG. 3. TCR Vβ interaction. The stereo view shows the complex between SPE-C (above,

green ribbon) and the TCR Vβ chain (below, magenta ribbon), determined by Sundberg et al

(21), with key SPE-C residues shown in red. Superimposed on SPE-C is a molecule of SPE-

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J (semi-transparent), showing how closely the two protein structures match. Also shown

(below, semi-transparent, under the Vβ structure) is the other molecule of the SPE-J dimer,

showing how both Vβ and dimerization use the same binding surface on SPE-J. SPE-J side

chains Tyr14, Phe77 and Arg181, shown to be important in both TCR binding and

dimerization are in blue.

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TABLES

Table 1. Relative molecular mass of SPE-J calculated from light scattering data Concentration (mg/mL) Mr from RHa (kDa) Cp/RHa (%)

1.0 23.3 27 2.0 33.4 25 4.0 44.2 19 5.5 54.6 14 6.0 52.5 17 10.0 61.1 14 aRH = hydrodynamic radius

Table 1. Data collection, refinement and model details SpeJ Zn-SpeJData collection Resolution (Å) 40 - 1.6 30 - 1.9Multiplicity 4.8 (4.8) 5.4 (5.3)Unique reflections 72162 44041Completeness (%) 99.4 (99.4) 99.9 (99.9)Rmerge (%) 5.5 (45.3) 6.4 (40.0)

I/σI 7.9 (1.9) 8.45 (1.9)

Refinement Resolution limits (Å) 1.75 2.0No. of reflections 51993 36793R (Rfree) (%) 20.9 (23.9) 21.8 (24.9)Protein atoms 5162 5162

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Ions - 3 Zn2+Water molecules 314 159

Geometry rmsd bond lengths (Å) 0.005 0.006rmsd bond angles (deg) 1.22 1.19% most favoured in 87.2 85.9Ramachandran plot

Table 3. TCR binding mutants of SPE-J

TCR interaction SPE-J Decrease inSPE-C H bond Vd

W SPE-J mutants mitogenecit

y

Y15 + + Y14 Y14A 10,000-foldT18 (+) + E17 E17A 0T20 (+) + I19 I19A 0R45 + + K44 K44A 10-fold

K46A 0 Y49 + - F48 F48A 0L78 - + F77 F77A 100-foldN79 + + T78 T78A 2-foldE178 - + S178 S178A 0R181 + - R181 R181Q 10,000-fold

(+) hydrogen bonds involving only main chain atoms.

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Supplementary data

Table 3. PCR primers used for mutagenesis of SpeJ pGEX.fw 5’-ACCATCCTCCAAAATCGG-3’pGEX.rev 5’-TCAGAGGTTTTCACCGTC-3’ SpeJ Y14A.fw 5’-CAATTAAATGCCGCATACGAAATCATAC-3’SpeJ Y14A.rev 5’-GGCATTTAATTGTAGCTTAACGTC-3’SpeJ E17A.fw 5’-CGCATACGCAATCATACCAGTAG-3’SpeJ E17A.rev 5’-GATTGCGTATGCGTAATTTAATTG-3’SpeJ I19A.fw 5’-CGAAATCGCACCAGTAGATTATACG-3’SpeJ I19A.rev 5’-CTGGTGCGATTTCGTATGCGTAAT-3’SpeJ K44A.fw 5’-CCAGTTATGCAAAGAAAAATTTTTCAG-3’SpeJ K44A.rev 5’-CTTTGCATAACTGGAAATATCAATAT-3’SpeJ K46A.fw 5’-TAAAAAGGCAAATTTTTCAGTTGATTC-3’SpeJ K46A.rev 5’-AAATTTGCCTTTTTATAACTGGAAATATC-3’SpeJ F48A.fw 5’-AAAAATGCTTCAGTTGATTCTGAG-3’SpeJ F48A.rev 5’-GAAGCATTTTTCTTTTTATAACTG-3’SpeJ F77A.fw 5’-CCGTACATAGCTACTCGTTATGATG-3’SpeJ F77A.rev 5’-AACGAGTAGCTATGTACGGAAGACC-3’SpeJ T78A.fw 5’-CATATTTGCTCGTTATGATGTTTATTAT-3’SpeJ T78A.rev 5’-CATAACGAGCAAATATGTACGGAAGAC-3’SpeJ Y83A.fw 5’-GATGTTGCTTATATATATGGTGGG-3’SpeJ Y83A.rev 5’-TATATAAGCAACATCATAACGAGTA-3’SpeJ S178A.fw 5’-CCTCAGCTAGTACAAGGAGTG-3’SpeJ S178A.rev 5’-GTACTAGCTGAGGTTGCATCATATAAG-3’SpeJ R181Q.fw 5’-TCTAGTACACAGAGTGATATTTTTAAA-3’SpeJ R181Q.rev 5’-CACTCTGTGTACTAGATGAGGTTGC-3’

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Edward N. BakerHeather M. Baker, Thomas Proft, Phillip D. Webb, Vickery L. Arcus, John D. Fraser and

dimerizationJ (SPE-J) can use a common binding surface for T-cell receptor binding and

Crystallographic and mutational data show that the streptococcal pyrogenic exotoxin

published online July 7, 2004J. Biol. Chem. 

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