Structural characterization of the sensor/transductor protein MecR1 of the Staphylococcus aureus β-lactam resistance systems.

HERMOSO, J.A.; FABBRI, C.; LLARRULL, L.I.

San Luis

Congreso; XLVIII Reunion Anual de la Sociedad Argentina de Biofisica; 2019

Sociedad Argentina de Biofisica

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a globally important pathogen that is resistant to all classes of β-lactam antibiotics and some strains are also resistant to glycopeptides, the last resort antibiotics used to treat MRSA infections. Resistance to β-lactam antibiotics in MRSA is due to the inducible expression of an accessory transpeptidase, PBP2a, with low affinity for most β-lactams and of the serin-β-lactamase PC1. The sensor/transducer proteins MecR1 and BlaR1 regulate the level of expression of PBP2a and PC1, respectively, in response to the presence of the β-lactam antibiotic. However, the intramolecular events that lead to activation of MecR1 and BlaR1 are poorly understood, and the search for inhibitors has been limited by the lack of high-resolution structural information on the full-length proteins. In order to gain insight into the molecular details of the activation of MecR1 we have evaluated the expression of full-length MecR1 (E205A mutant, which lacks autoproteolytic activity) as a fusion to the protein Mistic. We have succeeded in overexpressing Mistic-MecR1 in E. coli BL21 StarTM (DE3) membranes and we were able to purify it in detergent micelles to homogeneity using affinity chromatography. An assay with the fluorescent penicillin Bocillin-FL showed an active sensor domain in this recombinant protein. In addition, we have obtained polycrystals in a crystallogenesis screening assay. We attempted to optimize the crystallization condition but we have not achieved the formation of individual crystals yet. We are currently exploring our proposed mechanism of activation of MecR1 by β-lactams using site-directed mutagenesis in a S. aureus reporter strain and using photoactive amino acids.AcknowldegmentsANPCyT for the PhD fellowship awarded to CF. ASBMB for the PROLAB fellowship to CF. ANPCyT for grant PICT-2015-2521. CELFI fellowship to CF. Dr. Juan Hermoso for receiving me in his laboratory and teaching me practical knowledge about crystallography.