Understanding the structural and mechanistic details of protein-DNA interactions that lead to cellular defence against toxic metal ions in pathogenic bacteria can lead to new ways of combating their virulence....
Recently, site-directed Cu2+ labeling has emerged as an incisive biophysical tool to directly report on distance constraints that pertain to the structure, conformational transitions, and dynamics of proteins and nucleic acids. However, short phase memory times inherent to the Cu2+ labels limit measurable distances to 4–5 nm. In this work we systematically examine different methods to dampen electron–nuclear and electron–electron coupled interactions to decrease rapid relaxation. We show that using Cu2+ spin concentrations up to ca. 800 μM has an invariant effect on relaxation and that increasing the cryoprotectant concentration reduces contributions of solvent protons to relaxation. On the other hand, the deuteration of protein and solvent dramatically increases the duration of the dipolar modulated signal by over 6-fold to 32 μs. Based on this increase in signal longevity, distances up to 9 nm and beyond can potentially be measured with Cu2+ labels.
Metalloregulators bind and respond to metal ions by regulating the transcription of metal homeostasis genes. Copper efflux regulator (CueR) is a copper‐responsive metalloregulator that is found in numerous Gram‐negative bacteria. Upon Cu(I) coordination, CueR initiates transcription by bending the bound DNA promoter regions facilitating interaction with RNA polymerase. The structure of Escherichia coli CueR in presence of DNA and metal ion has been reported using X‐ray crystallography and cryo‐EM, providing information about the mechanism of action. However, the specific role of copper in controlling this transcription mechanism remains elusive. Herein, we use room temperature electron paramagnetic resonance (EPR) experiments to follow allosterically driven dynamical changes in E. coli CueR induced by Cu(I) binding. We suggest that more than one Cu(I) ion binds per CueR monomer, leading to changes in site‐specific dynamics at the Cu(I) binding domain and at the distant DNA binding site. Interestingly, Cu(I) binding leads to an increase in dynamics about 27 Å away at the DNA binding domain. These changes in the dynamics of the DNA binding domain are important for exact coordination with the DNA. Thus, Cu(I) binding is critical to initiate a series of conformational changes that regulate and initiate gene transcription. Broad audience statement The dynamics of metal transcription factors as a function of metal and DNA binding are complex. In this study, we use EPR spectroscopy to measure dynamical changes of Escherichia coli CueR as a function of copper and DNA binding. We show that copper controls the activation of the transcription processes by initiation a series of dynamical changes over the protein.
Oxidative stress is known to contribute to the progression of apoptosis. Staurosporine is a broad-spectrum inducer of apoptosis, but its mechanism of action is not well understood. The goal of the present work was to elucidate the role of glutathione and reactive oxygen species (ROS) in the execution of staurosporine-induced apoptosis. HeLa cells were treated with staurosporine at 1 μM for up to 4 h. The concentration of glutathione, generation of ROS, and activation of caspase-3 were measured. The introduction of staurosporine significantly decreased the concentration of cellular glutathione and increased the presence of ROS after 3 h. These findings were concurrent with the activation of caspase-3. Interestingly, pre-treatment of cells with N-acetylcysteine, a precursor of glutathione, and a thiol antioxidant failed to block the depletion of glutathione, generation of ROS, and activation of caspase-3. Collectively, these results suggest that the cellular redox status may be one of the critical factors of the apoptotic pathway leading to caspase-3 activation by staurosporine.
Mycobacterium tuberculosis is the causative agent of many strains of tuberculosis, as it is composed of an impenetrable, complex cell wall. The proteins active in the synthesis of the cell wall are mycolyl transferase antigens 85A, 85B, and 85C, encoded by genes fbpA, fbpB, and fbpC. Antigen 85C contains one cysteine residue. S-Glutathionylation is the formation of a mixed disulfide between a protein cysteine residue and glutathione (GSH), an abundant antioxidant molecule. It is a post-translational modification of cysteine residues which can occur under oxidative stress or physiological conditions. It is a known mechanism to regulate enzyme activity, signaling pathways, and the progression of diseases. By S-glutathionylation, the lone cysteine residue in antigen 85C is modified by biotinylated GSH ethyl ester to form a mixed disulfide. This modification results in a decrease in enzyme activity by 90%, representing a decrease in ability of the protein to synthesize the bacterial cell wall. Both the modification and the enzymatic activity of the protein are concentration dependent and can be reversed upon addition of a thiol reducing agent. The results provide a potential strategy for inhibiting the synthesis of the cell wall of M. tuberculosis by promoting oxidation of the lone cysteine residue. To our knowledge, this is a novel finding to demonstrate the modification of antigen 85C and the regulation of its activity by a physiological molecule. © 2018 IUBMB Life, 70(11):1111-1114, 2018.
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