Structural and functional characterization of short versions of BlaR1 and MecR1 of Staphylococcus aureus

LLARRULL, L.I.; MIHOVILCEVIC, D.

San Luis

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

Sociedad Argentina de Biofisica

The membrane proteins BlaR1 and MecR1 from the pathogen Staphylococcus aureus detect β-lactams and unleash a cascade of events that results in manifestation of resistance. Little is known about the intramolecular events that lead to activation of the cytoplasmic metallo-protease domain of these proteins. Full-length MecR1 and BlaR1 have not been crystallized so far, due to failed attempts to overexpress these two membrane proteins. Here we report the overexpression in E. coli and purification of two shorter versions of these sensor proteins. BlaR1JH1 is a shorter version of BlaR1 present in the β-lactam-resistant strain S. aureus JH1. BlaR1JH1 lacks the first three transmembrane helices and was overexpressed as a fusion to Mistic. MecR1-glz is the gluzincin domain of MecR1 and was expressed with a C-terminal His6x-tag. This domain contains the Zn(II) binding site of MecR1 with the mutation E205A (devoid of autoproteolytic activity). Mistic-BlaR1JH1 presented an active sensor domain that was irreversibly acylated by the fluorescent penicillin Bocillin-FL. MecR1-glz inserted into the membrane of E. coli, in accordance with our proposal of the presence of a reentering loop that functions as a membrane anchor. Using circular dichroism, we confirmed that MecR1-glz was structured in ASB-14 micelles, with a secondary structure composition in agreement with our bioinformatic model of MecR1. MecR1-glz has a single Trp residue in the reentering loop and anchoring to the membrane would protect it from the solvent. However, fluorescence quenching assays with KI showed that this Trp was solvent accessible in ASB-14 micelles. In conclusion, we have succeeded in purifying two shorter versions of the BlaR1 and MecR1 proteins. Initial characterization of MecR1-glz indetergent micelles indicates incompatibilities with our model in a membrane environment. Hence, we are now setting up the conditions to purify them in nanodiscs, so we can study the proteins in their native environment.AcknowldegmentsTo the National Research Council of Argentina for the Ph.D. fellowship. To ANPCyT for grant PICT-2015-2521.