Structural comparison of the exfoliative E toxin wild type and mutant of Staphylococcus aureus by X-ray crystallography and SAXS

Staphylococcus aureus is a commensal and opportunistic bacterium that can infect a variety of hosts, including humans and different species of cattle, represents a global public health problem, and causes enormous damage to the livestock industry. Exfoliative toxin E (ETE) is a virulence factor that facilitates the spread of the pathogen by inducing the generalized detachment of the granular layer of the epidermis in sheep. This serine protease is inactive in its native state. Occasional secondary interactions, which are still unknown in this system, and uncommon between serine proteinases induce specific hydrolysis of the peptide bond between Glu381-Gly382 of desmoglein 1 (Dsg1). A better understanding of the mechanism of action of ETs is necessary to develop strategies to inhibit the pathogen. This work seeks to elucidate the mechanisms and modes of action at the atomic level, in interactions with the substrate Dsg1. For this, the native and mutant ETE protein (Ser219Ala) were expressed in E. coli BL21 (DE3) -T1R. Subsequently, they were purified by gel filtration, on a Superdex® 75 10/300 GL column in 20 mM MES buffer pH 7.03, and pH 5.5. We analyzed the proteins with by circular dichroism, dynamic light scattering, and differential scanning calorimetry. its secondary structure, showing that both proteins have a predominance of random coil, in the particle size for the two proteins was 2.093 ± 0.3 (r.nm). Thermal unfolding experiments using circular dichroism and differential scanning calorimetry allowed us to establish the values of the melting temperature (49.0 ± 1.4), the calorimetric enthalpy (220 ± 50), and the Van't Hoff enthalpy (270 ± 70). Crystals obtained from the proteins were diffracted in the MANACÁ line of LNLS, from Campinas-Brazil. The final resolution for ETEwt was 1.6 Å and 2.4 Å for ETEmut, both forming dimers. Investigations by low angle X-ray scattering (SAXS) data indicated the presence of oligomers in the system, depending on the concentration of the sample.