Architecture of MecR1: a membrane-embedded metalloprotease domain poised to receive information fromthe extracelular sensor domain

GIANNINI, E.; ABRIATA, L.A.; LLARRULL, L.I.; BELLUZO, B.S.; DAL PERARO, M.

Buenos Aires

Congreso; Joint Meeting of Bioscience Societies; 2017

Abstract: The signal transducer protein MecR1 from methicillin resistant Staphylococcus aureus regulates the expression of PBP2a, a protein that is not inhibited by clinical concentrations of most β-lactam antibiotics. In this study we aimed at identify a structural model that present a better comprehension of the signal transduction mediated by MecR1, which is an attractive target for drug development. By integrating homology modeling, residue co-evolution analysis, dynamic simulations and docking, we generated a model for the full length MecR1 protein. This model was used to design a series of constructions to further evaluate the topology in-vitro. Fluorescence spectroscopy and Proteinase K susceptibility assays in MecR1.eGFP truncated fusions and TEV peptidase susceptibility assays in MecR1.E205A.TEV insertions were carried out. As a whole, these data allowed us to corroborate the orientation of most transmembrane helixes, it showed that the peptide S63-T102 is extracellular (and not a TM helix) and that Loop 4 in the metalloprotease domain N194 to D213 has low flexibility, which could be due to compaction or membrane interaction. To further evaluate if the effector domain has membrane localization, the cytoplasmic domain (cytMecR1), was expressed in E. coli, and we found it was membrane embedded. These results support the existence of a reentering alpha-helix in the metalloprotease domain that tightly anchors this domain to the membrane, in accordance with our computational model. In conclusion, we presented a model for full length MecR1 in which the metalloprotease domain is embedded in the membrane, defining a hydrophilic chamber. A reentering loop connecting the metal ligands tightly anchors the metalloprotease domain to the membrane (even in the absence of the rest of the TM helixes) and reaches to the outer leaflet of the membrane where it is posed to interact with the sensor domain loops that show altered mobility upon antibiotic binding.