The Dynamic Plasticity of P. aeruginosa CueR Copper Transcription Factor upon Cofactor and DNA Binding.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E. coli CueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However, P. aeruginosa CueR, which shows high sequence similarity to E. coli CueR, has been less studied. Here, we applied room-temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics of P. aeruginosa CueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed that P. aeruginosa CueR is less dynamic than the E. coli CueR protein and exhibits much higher sensitivity to DNA binding as compared to its E. coli CueR homolog. Moreover, a difference in dynamical behavior was observed when P. aeruginosa CueR binds to the copZ2 DNA promoter sequence compared to the mexPQ-opmE promoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ-OpmE proteins in P. aeruginosa. Overall, such comparative measurements of protein-DNA complexes derived from different bacterial systems reveal insights about how structural and dynamical differences between two highly homologous proteins lead to quite different DNA sequence-recognition and mechanistic properties.

Double Quantum Coherence ESR at Q-Band Enhances the Sensitivity of Distance Measurements at Submicromolar Concentrations.

Recently, there have been remarkable improvements in pulsed ESR sensitivity, paving the way for broader applicability of ESR in the measurement of biological distance constraints, for instance, at physiological concentrations and in more complex systems. Nevertheless, submicromolar distance measurements with the commonly used nitroxide spin label take multiple days. Therefore, there remains a need for rapid and reliable methods of measuring distances between spins at nanomolar concentrations. In this work, we demonstrate the power of double quantum coherence (DQC) experiments at Q-band frequencies. With the help of short and intense pulses, we showcase DQC signals on nitroxide-labeled proteins with modulation depths close to 100%. We show that the deep dipolar modulations aid in the resolution of bimodal distance distributions. Finally, we establish that distance measurements with protein concentrations as low as 25 nM are feasible. This limit is approximately 4-fold lower than previously possible. We anticipate that nanomolar concentration measurements will lead to further advancements in the use of ESR, especially in cellular contexts.

Allostery-driven changes in dynamics regulate the activation of bacterial copper transcription factor.

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.

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator.

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. Herein, we examine the Copper Efflux Regulator (CueR) protein, a transcription factor which interacts with DNA to generate proteins that ameliorate excess free Cu(i). We exploit site directed Cu(ii) labeling to measure the conformational changes in DNA as a function of protein and Cu(i) concentration. Unexpectedly, the EPR data indicate that the protein can bend the DNA at high protein concentrations even in the Cu(i)-free state. On the other hand, the bent state of the DNA is accessed at a low protein concentration in the presence of Cu(i). Such bending enables the coordination of the DNA with RNA polymerase. Taken together, the results lead to a structural understanding of how transcription is activated in response to Cu(i) stress and how Cu(i)-free CueR can replace Cu(i)-bound CueR in the protein-DNA complex to terminate transcription. This work also highlights the utility of EPR to measure structural data under conditions that are difficult to access in order to shed light on protein function.

dHis-troying Barriers: Deuteration Provides a Pathway to Increase Sensitivity and Accessible Distances for Cu2+ Labels.

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.

Role of Glutathione Depletion and Reactive Oxygen Species Generation on Caspase-3 Activation: A Study With the Kinase Inhibitor Staurosporine.

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.

Regulation of antigen 85C activity by reversible S-glutathionylation.

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.