Streptococcal Pyrogenic Exotoxin A1
Joel Beckett '08 and Andy Boylan '08
Edited by Daniel Barich '05
Contents:
I. Introduction
Streptococcus pyogenes (group A streptococcus) is a virulent organism
that causes scarlet fever, rheumatic fever and streptococcal toxic shock syndrome
(STSS). While modern antibiotics have effectively combated the two former illnesses,
STSS seems to be occurring with increasing frequency. STSS is often caused by
a bacterial infection of a minor skin lesion or bruise and is characterized
by rash, fever, hypotension and multiorgan failure; 30% to 70% of infected individuals
die in spite of aggressive modern medicine (Stevens, 1999).
Streptococcal pyrogenic exotoxin A1 (SpeA1) is a superantigen commonly
isolated from streptococcal strains infecting individuals with STSS. Superantigens
are a family of proteins able to simultaneously bind major histocompatibility
complex (MHC) class II molecules and the T-cell receptor (TcR). Superantigens
bind outside the MHC-TcR binding groove occupied by conventional antigens.
(Papageorgiou and Acharya 1997). Such binding specifically stimulates Vß
expressing T-cells, mitigating a massive release of cytokines by lymphocytes
and monocytes. (Papageorgiou et al, 1999). While a normal antigen stimulates
0.001% of T-cells, a superantigen may stimulate nearly 20% (Papageorgiou and
Acharya, 1997). This catastrophic buildup of cytokines leads to acute shock,
a symptom typical of STSS.
II. General Structure
SpeA1 crystallizes in an asymmetric unit of four individual
complete molecules.
Each monomer of SpeA1 is comprised of a single polypeptide
chain of 221 amino acids. Similar to other superantigens, the N terminal domain
contains a ß-barrel tertiary structure called the 'oligosaccharide
/ oligonucleotide fold '
,
which is comprised of five ß sheets. ß strands 2 and 3 are antiparallel,
while the ß1 strand runs parallel to 5 and anti-parallel to 4. A long
α-helix
runs down the center of the protein connecting the N and C terminal domains.
ß sheets 6, 7, 12, 9, and 10 comprise a ß
grasp motif
characteristic of C terminal domains in the superantigen family (Papageorgiou
et al, 1999).
Each molecule of SpeA1 may potentially bind a zinc
ion using a binding site located at the interface between the N and C
terminal domains
.
Glu 33, Asp 77, His
106, and His 110 move slightly to coordinate
the binding of the single zinc ligand (Baker et al, 2001).
SpeA1 also contains an intramolecular disulfide
bond between Cys-87 and Cys-98
.
This bond connects the ß4 and ß5 sheets at the top of the N-terminal ß-barrel
and forms a 'flexible loop'
.
Located on this loop is Cys-90
which is involved in dimerization (Papageorigou et al, 1999).
III. Dimerization
The dimeric structure forms through the creation of a
disulfide bond between two identical monomers.
Cysteines located on the flexible loop (residues
87-98) at position 90 create an intermolecular disulfide
bond, providing the foundation for dimeric quaternary structure
(Papageorgiou et al, 1999). Formation of this disulfide linkage requires the
movement of the flexible loop between 1.62 and 1.96 Å from its position
in the native monomeric structure. While the individual amino acid sequence
of this loop may vary from one molecule to another, the Cys-90 is conserved,
indicating its importance in the establishment of the dimer (Baker et al, 2004).
IV. MHC Class II Binding
Toxins in the streptococcal family each exhibit a high affinity for binding
a particular subset of MHC class II molecules. The SpeA1 toxin possesses a high
affinity for the HLA-DQ type MHCs. Binding of these molecules occurs through
one of two binding sites (Papageorgiou, 1999).
The generic MHC binding site
provides
a non-ligand mediated method of MHC binding. Mutational studies have identified
the following amino acids as crucial to generic MHC class II binding in SpeA1:
Leu 42, Asp 45, Leu
46, Ile 47, Tyr 48,
and Tyr 83
(Papageorgiou et al, 1999).
Dimerization appears to block the generic MHC binding site, sequestering
the essential residues within the interface between the two monomers
.
If this steric hinderance of MHC binding does occur, then zinc-mediated MHC
binding would be the only available method of TcR-MHC complex formation. (Baker
et al, 2001)
Ligand mediated binding of MHC class II molecules occurs at the zinc
binding site
, located in the region near the N and C terminal domains.
In SpeA1, mutagenesis studies have concluded that the zinc site is
crucial to MHC binding, as mutations within any of the key residues significantly
decreases ability to bind MHC II molecules, even to the point of nonexistent
affinity (Hartwig and Fleischer, 1993). Additionally, in other protein members
of the superantigen family, metal ion binding sites have been shown to not only
influence MHC binding, but also dimer formation and thermostability (Baker et
al, 2001).
V. T-cell Receptor Binding
SpeA1 binds T-cells expressing the Vß subset of
T-cell receptor proteins, specifically Vß 2.1, 12.2, 14.1, and 15.1 (Kline
and Collins, 1997). The variability within the structures of these different
receptors indicates that the residue used for specific receptor recognition
varies across a range of amino acids (Papageorgiou et al, 1999). The TcR
binding site is located in two α helices as well as the flexible
disulfide loop
.
This binding site is adjacent to the MHC II binding site
,
providing further structural support for the conclusion that superantigens bind
both the MHC II and TcR molecules outside of the standard binding groove motif.
A depiction of the complex can be seen here: MHC-TcR
SpeA1 Complex
Similar to the MHC II binding site, dimerization appears to sterically
inhibit the access of TCR molecules to the requisite binding site
.
Given that only one TcR binding site exists within the protein, such dimeric
structure would appear to prevent MHC-TcR complex formation. As mentioned above,
experimental data contradicts this conclusion, indicating equal viability of
both the monomeric and dimeric forms. An explanation for this apparent discrepancy
lies in the postulated flexibility of the disulfide loop, a quality not reflected
in a rigid protein crystal structure (Baker et al, 2004). Such flexibility of
the loop and the dimer-mediating residues it includes allows for novel orientations
of both the TcR and MHC II binding sites. The prevalence of such variable orientations
in general toxin structure increase the possibility of binding of multiple MHC
and TcR molecules, imparting greater potency on the dimeric form.
VI. Future of SpeA1
Recent years have witnessed an increase in the frequency of infections
due to Streptococcus pyrogenes and deaths from STSS. The SpeA1 toxin is released
by Streptococcus, stimulating a response on the immunocellular level. Research
into the structure of SpeA1 and its ability to simulanteously bind MHC II and
TcR molecules has revealed a dynamic protein potent in both monomeric and dimeric
forms.
While evidence from the crystal structure indicates that dimeric binding
blocks formation of MHC-TcR complex, recent experimental data contradicts this
conclusion. Both forms have been shown to exhibit equally potent activity in
vitro. One prediction even postulates a greater potency of the dimeric form
in vivo (Baker et al, 2004). This discrepancy is explained through the flexibility
of the linker region. Linker elasticity allows the protein to take a number
of different orientations, opening multiple binding sites to both MHC and TcR
molecules. In order understand and combat this protein, further research is
needed to elucidate the true nature of TcR-MHC complex formation in monomeric
and dimeric forms.
Prepare for Blastoff!
VII. References
[1] Baker, Matthew D., Inessa Gendlina,
Carleen M. Collins, and K. Ravi Acharya. 2004. Crystal structure of a dimeric
form of streptococcal pyrogenic exotoxin A (SpeA1). Protein Science 13:2285-2290.
[2] Baker, Matthew, Delia M. Gutman, Anastassios
C. Papageorgiou, Carleen M. Collins, and K. Ravi Acharya. 2001. Structural features
of a zinc binding site in the superantigen streptococcal pyrogenic exotoxin
A (SpeA1): Implications for MHC class II recognition. Protein Science
10:1268-1273.
[3] Hartwig, U.F., and B. Fleischer. 1993.
Mutations affecting MHC class II binding of the superantigen streptococcal erythrogenic
toxin A. International Immunology 5: 869-875.
[4] Kline, J. Bradford, and Carleen M. Collins.
1997. Analysis of the interaction between the bacterial superantigen streptococcal
pyrogenic exotoxin A (SpeA) and the human T-cell receptor. Molecular Microbiology
24:191-202.
[5] Papageorgiou, Anastassios C., Carleen
M. Collins, Delia M. Gutman, J. Bradford Kline, Susan M. O'Brien, Howard S.
Tranter, and K. Ravi Acharya. 1999. Structural basis for the recognition of
superantigen streptococcal pyrogenic exotoxin A (SpeA1) by MHC class II molecules
and T-cell receptors. The EMBO Journal 18:9-21.
[6]Papageorgiou, Anastassios C., and K.
Ravi Acharya. 1997. Superantigens as immunomodulators: recent structural insights.
Structure 5: 991-996.
[7] Stevens, Dennis L. 1995. Streptococcal
Toxic-Shock Syndrome: Spectrum of Disease, Pathogenesis, and New Concepts in
Treatment. Emerging Infectious Diseases 1:69-78.