Matthew Douglas Baker

Superantigens: structure-function relationships.

Superantigens are a class of highly potent immuno-stimulatory molecules produced by Staphylococcus aureus and Streptococcus pyogenes. These toxins possess the unique ability to interact simultaneously with MHC class II molecules and T-cell receptors, forming a trimolecular complex that induces profound T-cell proliferation. The resultant massive cytokine release causes epithelial damage and leads to capillary leak and hypotension. The staphylococcal superantigens are designated staphylococcal enterotoxins A, B, C (and antigenic variants), D, E, and the recently discovered enterotoxins G to Q, and toxic shock syndrome toxin-1. The streptococcal superantigens include the pyrogenic exotoxins A (and antigenic variants), C, G-J, SMEZ, and SSA. Superantigens are implicated in several diseases including toxic shock syndrome, scarlet fever and food poisoning; and their function appears primarily to debilitate the host sufficiently to permit the causation of disease. Structural studies over the last 10 years have provided a great deal of information regarding the complex interactions of these molecules with their receptors. This, combined with the wealth of new information from genomics initiatives, have shown that, despite their common molecular architecture, superantigens are able to crosslink MHC class II molecules and T-cell receptors by a variety of subtly different ways through the use of various structural regions within each toxin.

Structural Features of a Zinc Binding Site in the Superantigen Strepococcal Pyrogenic Exotoxin a (Spea1): Implications for Mhc Class II Recognition.

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 Å resolution. The protein crystallizes in the orthorhombic space group P21212, 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.

Crystal structure of a dimeric form of streptococcal pyrogenic exotoxin A (SpeA1).

Streptococcal pyrogenic exotoxin A (SpeA1) is a bacterial superantigen associated with scarlet fever and streptococcal toxic shock syndrome (STSS). SpeA1 is found in both monomeric and dimeric forms, and previous work suggested that the dimer results from an intermolecular disulfide bond between the cysteines at positions 90 of each monomer. Here, we present the crystal structure of the dimeric form of SpeA1. The toxin crystallizes in the orthorhombic space group P212121, with two dimers in the crystallographic asymmetric unit. The final structure has a crystallographic R‐factor of 21.52% for 7248 protein atoms, 136 water molecules, and 4 zinc atoms (one zinc atom per molecule). The implications of SpeA1 dimer on MHC class II and T‐cell receptor recognition are discussed.

Influence of Naturally-Occurring 5'-Pyrophosphate-Linked Substituents on the Binding of Adenylic Inhibitors to Ribonuclease A: An X-Ray Crystallographic Study.

Ribonuclease A is the archetype of a functionally diverse superfamily of vertebrate‐specific ribonucleases. Inhibitors of its action have potential use in the elucidation of the in vivo roles of these enzymes and in the treatment of pathologies associated therewith. Derivatives of adenosine 5′‐pyrophosphate are the most potent nucleotide‐based inhibitors known. Here, we use X‐ray crystallography to visualize the binding of four naturally‐occurring derivatives that contain 5′‐pyrophosphate‐linked extensions. 5′‐ATP binds with the adenine occupying the B2 subsite in the manner of an RNA substrate but with the γ‐phosphate at the P1 subsite. Diadenosine triphosphate (Ap3A) binds with the adenine in syn conformation, the β‐phosphate as the principal P1 subsite ligand and without order beyond the γ‐phosphate. NADPH and NADP+ bind with the adenine stacked against an alternative rotamer of His119, the 2′‐phosphate at the P1 subsite, and without order beyond the 5′‐α‐phosphate. We also present the structure of the complex formed with pyrophosphate ion. The structural data enable existing kinetic data on the binding of these compounds to a variety of ribonucleases to be rationalized and suggest that as the complexity of the 5′‐linked extension increases, the need to avoid unfavorable contacts places limitations on the number of possible binding modes.

CHAPTER 53 – Comparative three-dimensional structure of bacterial superantigenic toxins

From the first three-dimensional structure of a bacterial superantigen to the complexes of these toxins with their receptors the complete archaeology of these molecules has been mapped and a wealth of information has been obtained. The ability to assign specific functions to discrete regions of the toxin and to assess the roles of individual amino acid residues by a combination of mutagenesis and structural techniques has enabled detailed comparisons of the entire bacterial superantigen family of proteins. Superantigens are powerful T cell stimulatory molecules produced primarily by Staphylococcus aureus and Streptococcus pyogenes. The action of these toxins as SAgs can be attributed to their ability to cross-link MHC class II molecules and T cell receptors to form a trimolecular complex. The number of known bacterial proteins with superantigenic properties and/or high homology with known SAgs has grown considerably over the last decade. Staphylococcal enterotoxins (SEs), A, B, Cl-3, D, E, H, I, J; toxic shock syndrome toxin-1 (TSST-1), the streptococcal pyrogenic exotoxins (Spes) A, C, H; and streptococcal mitogenic exotoxin SME-Z 2 and streptococcal superantigen (SSA) are the most well studied bacterial SAgs to date. Other pathogens, such as Mycoplasma arthritidis and Yersinia enterocolitica have also been shown to secrete superantigenic proteins and the characterization of these toxins has recently been helped by the elucidation of their crystal structures.