Characterization of the Transcriptional Regulator MecI from Staphylococcus aureus

BLAZQUEZ, B.; LLARRULL, L.I.; LUQUE-ORTEGA, J.R.; ALFONSO, C.; BOGGESS, B.; MOBASHERY, S.

Indianapolis

Congreso; 246th American Chemical Society National Meeting & Exposition; 2013

Beta-Lactam antibiotics are becoming less effective as therapeutics in treatment of staphylococcal infections as resistance to them increases. Resistance is mediated by a beta-lactamase (encoded by blaZ) that hydrolyzes penicillins and a penicillin-binding protein (PBP2a, encoded by mecA), which is not modified effectively by these antibiotics. The 14.8-kDa MecI protein represses transcription of mecA and its threedimensional structure reveals a dimer of two intimately intertwining dimerization domains, held together by a hydrophobic core, and two independent winged helix domains, each of which binds a palindromic DNA-operator half site. The mec operator consists of a single 30-bp palindrome with two 15-bp half-sites. We provide insight into the interactions of the MecI protein and the mec operator by a gel retardation assay. Equilibrium sedimentation studies provided the dissociation constant for the monomer-dimer equilibrium. Fluorescence anisotropy experiments have shown that binding of MecI to mec promoter is different depending on protein concentration. The anisotropy data has been fit using equations derived for different binding models, taking into account the possibility of binding of MecI as a monomer and/or dimer, taking into account the in vivo MecI concentrations, which were evaluated.