Structural and functional characterization of the sensor/transducer MecR1 protein of Staphylococcus aureus

MIHOVILCEVIC, D.; FABBRI, C.; SUAREZ, I.P.; HERMOSO, J.A.; LLARRULL, L.I.

Congreso; XLIX Reunión Anual de la Sociedad Argentina de Biofísica; 2021

Sociedad Argentina de Biofísica

Methicillin resistant Staphylococcus aureus (MRSA) is a pathogen that poses a worldwide threat. Resistance to b-lactams in MRSA is regulated by the membrane proteins BlaR1 and MecR1. We are interested in unveiling how b-lactams activate these metalloproteases, resulting in manifestation of resistance. Mechanistic and structural information of these sensor proteins is needed to rationally design compounds capable of blocking signal transduction that could be used in combination therapies with currently available β-lactam antibiotics. We have informed a model for full-length MecR1 combining modeling, experimental mapping of transmembrane topology, the X-Ray structure of the sensor domain and molecular simulations. However, no high-resolution models of full-length MecR1 or BlaR1 are available. In this work we intended to structurally and functionally characterize full-length MecR1. We have been able to over-express full-length MecR1 (E205A mutant) as a fusion to Mistic in E. coli BL21 StarTM (DE3) membranes. Furthermore, we have purified it by affinity and size-exclusion chromatography. To determine the homogeneity and oligomerization state of Mistic-MecR1 we have performed SEC-MALS and AUC experiments, which showed two oligomerization states in detergent micelles: a monomer (major species) and a dimer. Both species presented an active sensor domain that was irreversibly acylated by the fluorescent penicillin Bocillin-FL. We also performed Circular Dichroism spectra in different conditions. The protein was thermostable and could only be denatured in the presence of SDS and urea. We carried out crystallization assays, but we only obtained poly-crystals that could not be optimized. We concluded that Mistic-MecR1 is not a homogeneous sample, as it is in equilibrium between monomer and dimer, which might be interfering with crystallization. We will hence explore the possibility of characterizing Mistic-MecR1 using Cryogenic electron microscopy (cryo-EM).AcknowledgmentsD.M. and C.F. are Ph.D. Fellows of CONICET. I.P.S. and L.I.L. are Staff members of CONICET. This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) to L.I.L (PICT-2015-2521 and PICT-2018-3362) and from Instruct-Eric (PID 8070).