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Structural features of a zinc binding site in the superantigen strepococcal pyrogenic exotoxin A (SpeA1):
M Baker1, D M Gutman, A C Papageorgiou
1Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.
Abstract:
Streptococcal pyrogenic exotoxin A (SpeA) is produced by Streptococcus pyogenes, and has been associated with severe infections such as scarlet fever and Streptococcal Toxic Shock Syndrome (STSS). In this study, the crystal structure of SpeA1 (the product of speA allele 1) in the presence of 2.5 mM zinc was determined at 2.8 A resolution. The protein crystallizes in the orthorhombic space group P2(1)2(1)2, with four molecules in the crystallographic asymmetric unit. The final structure has a crystallographic R-factor of 21.4% for 7,031 protein atoms, 143 water molecules, and 4 zinc atoms (one zinc atom per molecule). Four protein ligands-Glu 33, Asp 77, His 106, and His 110-form a zinc binding site that is similar to the one observed in a related superantigen, staphylococcoal enterotoxin C2. Mutant toxin forms substituting Ala for each of the zinc binding residues were generated. The affinity of these mutants for zinc ion confirms the composition of this metal binding site. The implications of zinc binding to SpeA1 for MHC class II recognition are explored using a molecular modeling approach. The results indicate that, despite their common overall architecture, superantigens appear to have multiple ways of complex formation with MHC class II molecules.
Insights
The crystal structure of Streptococcal pyrogenic exotoxin A (SpeA1) bound to zinc was determined. This reveals a zinc-binding site crucial for understanding SpeA1
Area of Science:
- Structural biology
- Molecular microbiology
- Immunology
Background:
- Streptococcal pyrogenic exotoxin A (SpeA) is a key virulence factor from Streptococcus pyogenes.
- SpeA is associated with severe diseases, including scarlet fever and Streptococcal Toxic Shock Syndrome (STSS).
- Understanding SpeA's structure and function is critical for developing therapeutic strategies.
Purpose of the Study:
- To determine the high-resolution crystal structure of SpeA1 in complex with zinc.
- To characterize the zinc-binding site within SpeA1.
- To explore the implications of zinc binding for SpeA1's interaction with MHC class II molecules.
Main Methods:
- X-ray crystallography was used to determine the crystal structure of SpeA1 at 2.8 Å resolution.
- Site-directed mutagenesis was employed to generate mutants lacking key zinc-binding residues.
- Zinc-binding affinity assays and molecular modeling were utilized to analyze the metal-binding site and its functional implications.
Main Results:
- The crystal structure revealed a zinc-binding site formed by Glu33, Asp77, His106, and His110, similar to that in staphylococcal enterotoxin C2.
- Mutant toxins showed reduced affinity for zinc, confirming the identified metal-binding residues.
- Molecular modeling suggested that zinc binding may influence SpeA1's interaction with MHC class II molecules, despite conserved superantigen architectures.
Conclusions:
- The study elucidates the structural basis of zinc binding in SpeA1, identifying key residues involved in metal coordination.
- Zinc binding represents a potential regulatory mechanism for SpeA1 function and its interaction with the host immune system.
- The findings contribute to understanding the diverse mechanisms by which superantigens engage with MHC class II molecules.