Architecture of MecR1 of Staphylococcus aureus: clues to the signal transduction mechanism that unleashes resistance to β-lactams

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

Parana

Congreso; LIV Reunion Anual de la Sociedad Argentina de Bioquimica y Biologia Molecular; 2018

Sociedad Argentina de Bioquimica y Biologia Molecular

Resistance to beta-lactam antibiotics in the pathogen Staphylococcus aureus is due to the inducible expression of two enzymes: the serine-b-lactamase PC1 and the transpeptidase PBP2a, which is not inhibited by clinical concentrations of most of these antibiotics. The transmembrane sensor/signal transducer proteins MecR1 and BlaR1 detect the presence of the antibiotic and regulate the expression of the genes that result in manifestation of resistance. Beta-Lactam-acylation of the extracellular sensor domain of MecR1/BlaR1 is believed to be the initial event that unleashes activation of their metalloprotease domain through a yet inscrutable signal-transduction mechanism. The activated metalloproteases degrade the DNA-binding proteins MecI/BlaI. The latter are repressors of transcription of the genes of the operons. Hence, their degradation results in expression of PC1 and PBP2a. The mechanism of signal transduction used by MecR1 and BlaR1 is of great interest given that they are possible targets for the design of inhibitors that can restore the effectiveness of beta-lactam antibiotics for the treatment of S. aureus infections. The complete proteins or, for that matter, the transmembrane/metalloprotease domains of MecR1 and BlaR1 remain not amenable for structural studies. In this study we combined homology modeling and co-evolution-based de novo structure prediction to generate a model of the transmembrane domain of MecR1. This model was tested through an experimental mapping of the topology of the loops of this domain in E. coli, using fusions to eGFP and Proteinase K/TEV-protease susceptibility assays. The model suggested that the gluzincin core of the metalloprotease domain would be anchored to the cytoplasmic side of the membrane by a reentrant helix, hypothesis that we confirmed by expression of the isolated gluzincin core in E. coli membranes. Finally, we docked the structure of the sensor domain of MecR1 into our model of its metalloprotease domain, guided by interactions reported by NMR between the sensor domain of the homologous protein BlaR1 and an extracellular loop of the transmembrane domain. Integration of all these results allow us to put forward a model for full-length MecR1 where the metalloprotease domain defines a hydrophilic transmembrane chamber in whose floor the zinc site sits, sealed on the periplasmic side by the sensor domain. A reentrant helix anchors the gluzincin core of the metalloprotease domain to the cytoplasmic side of the membrane and is poised to interact with the sensor domain. This model accounts for all the available biochemical and structural information on this class of sensors and gives the first insights into the way the presence of the antibiotic modulates the activity of the membrane-embedded domain.