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Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity.

Exfoliative toxins produced by certain strains of Staphylococcus hyicus mediate exudative epidermitis in pigs. In this study the genes coding for four different exfoliative toxin from S. hyicus (ExhA, ExhB, ExhC, and ExhD) were cloned and sequenced. The coding sequence of the four toxin genes ranged from 816 to 834 bp. The amino acid sequences of these four toxins were homologous to the earlier described exfoliative toxins SHETB from S. hyicus and ETA, ETB, and ETD from Staphylococcus aureus. The homology between the S. hyicus toxins was at the same level as the homology to the exfoliative toxins from S. aureus. The toxins showed similarity to serine proteases, including preservation of the catalytic tract in ExhA, ExhB, and ExhC. However, in ExhD, Asp in the putative catalytic tract was replaced with Glu. The recombinant toxins could be expressed in Escherichia coli, and three of the four toxins were recognized by monoclonal antibodies raised against native exfoliative toxins.

Staphylococcal aureus epidermolytic toxins (ETs) A and B are responsible for the induction of staphylococcal scalded skin syndrome, a disease of neonates and young children. The clinical features of this syndrome vary from localized blisters to severe exfoliation affecting most of the body surface. Comparison of the crystal structures of two subtypes of ETs-rETA (at 2.0 A resolution), rETB (at 2.8 A resolution), and an active site variant of rETA, Ser195Ala at 2.0 A resolution has demonstrated that their overall topology resembles that of a "trypsin-like" serine protease, but with significant differences at the N- and C-termini and loop regions. The details of the catalytic site in both ET structures are very similar to those in glutamate-specific serine proteases, suggesting a common catalytic mechanism. However, the "oxyanion hole," which is part of the catalytic sites of glutamate specific serine proteases, is in the closed or inactive conformation for rETA, yet in the open or active conformation for rETB. The ETs contain a unique amphipathic helix at the N-terminus, and it appears to be involved in optimizing the conformation of the catalytic site residues. Determination of the structure of the rETA catalytic site variant, Ser195Ala, showed no significant perturbation at the active site, establishing that the loss of biological and esterolytic activity can be attributed solely to disruption of the catalytic serine residue. Finally, the crystal structure of ETs, together with biochemical data and mutagenesis studies, strongly confirms the classification of these molecules as "serine proteases" rather than "superantigens."

With a view to obtaining a better understanding of the structural determinants of P1 glutamate specificity in glutamate-specific endopeptidases (GSEs), the active sites and specificity pockets of such enzymes from Bacillus licheniformis (gse-bl), Bacillus subtilis (mpr) and Staphylococcus aureus (v8 protease) were modelled. This approach was extended to the epidermolytic toxins (ETs), responsible for the staphylococcal scalded skin syndrome. We identify a canonical structure for the S1 subsite, composed of H213 and T190, both of which we predict to interact directly with the P1 glutamate. The possible importance of R30 (for gse-bl and mpr) and of the N-terminus (for gse-bl, mpr and v8 protease) was also examined. In the case of mpr, a G193C substitution is predicted to participate in a novel disulphide bridge which stabilizes C193 in such a way as to maintain the oxyanion hole. In v8, the loss or substitution of several important structural components around D102 of the catalytic triad probably explains its reduced catalytic efficiency in comparison with other GSEs. In the case of the epidermolytic toxins K216 may be important for the previously reported phospholipase C-like activity, since the model predicts that it may stabilize the negative charge on the phosphonyl group.

The substitution of the serine 195 residue of staphylococcal exfoliative toxin A by a cysteine residue led to a biologically inactive protein. This result is consistent with the hypothesis that exfoliative toxin A could be a protease or a lipase. However, no protease or lipase activity was detected with the native toxin.