Agradecimentos: We thank Yves Le Loir for the donation of pD441/ETE expression vector. J.E.H.G. and R.K.A. acknowledge the National Laboratory for Scientific Computing (LNCC/MCTI, Brazil) for providing HPC resources of the SDumont supercomputer, which have contributed to the research results reported within this work (http://sdumont.lncc.br, accessed date: 26 May 2022). This research used facilities of the Brazilian Synchrotron Light Laboratory (LNLS), part of the Brazilian Center for Research in Energy and Materials (CNPEM), a private nonprofit organization under the supervision of the Brazilian Ministry for Science, Technology, and Innovations (MCTI). The W01B-MX2 beamline staff is acknowledged for their assistance during experiments 20190086. This research was funded by the Sao Paulo Research Foundation (FAPESP), Grants: 2020/13921-0, 2020/10214-1, 2018/07977-3, 2020/08615-8 and CNPq Grant 309940/2019-2 Ver menos

Abstract: 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 Ver menos