Gevorkyan-Airapetov, L. | Search bioRxiv

bioRxiv2022

An in cell site-specific labeling methodology reveals conformational changes of proteins in bacteria

Gaining new structural information on proteins in their native cellular environments will shed light on many enzymatic reaction mechanisms and encourage the development of new therapeutic approaches. During the last decade, in cell electron paramagnetic resonance (EPR) spectroscopy experiments have provided high-resolution data on conformational changes of proteins within the cell. However, one of the major obstacles of EPR spectroscopy is the spin-labeling process, which until now was performed only outside the cellular environment (i.e., exogenously). The spin-labeled protein is then injected into the cell, which limits the protein size and the cellular system that can be used. Here, we describe a new spin-labeling approach that can be applied to over-expressed proteins in Escherichia coli (i.e., endogenously). This approach uses a Cu(II) ion bound to a ligand, which has high affinity to a dHis site in the protein of interest. The presence of a nearby 19F-phenylalanine residue can be exploited to verify that the Cu(II)-ligand indeed bound to the protein target. This new methodology allows for the study of any protein, regardless of size or the cellular system used.

biophysics↗

bioRxiv2019

Inhibiting the copper efflux system in microbes as a novel approach for developing antibiotics

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living cells, but it can be toxic in excess. Both eukaryotic and prokaryotic cells have developed distinct copper regulation systems to prevent its toxicity. Therefore, selectively targeting the prokaryotic copper regulation system might be an initial step in developing next-generation antibiotics. One such system is the Gram-negative bacterial CusCFBA efflux system. CusB is a key protein in this system and was previously reported to play an important role in opening the channel for efflux via significant structural changes upon copper binding while also controlling the assembly and disassembly process of the entire channel. In this study, we aimed to develop novel peptide copper channel blockers, designed by in silico calculations based on the structure of CusB. Using a combination of magnetic resonance spectroscopy and various biochemical methods, we found a lead peptide that promotes copper-induced cell toxicity. Targeting copper transport in bacteria has not yet been pursued as an antibiotic mechanism of action. Thus, our study lays the foundation for discovering novel antibiotics.\n\nAuthor SummaryHerein, we apply a novel approach for the development of a new generation of antibiotics based on copper toxicity. In cells, copper ions are double-edge swords. On the one hand, various enzymes depend on them as cofactors for catalysis, but on the other hand, they are highly toxic. Thus, cells have developed sophisticated regulation systems to very precisely control copper concentration. Prokaryotic organisms are more sensitive to copper than eukaryotic systems, and therefore they employ additional copper transporters that have no homology in the eukaryotic cells in general and specifically in the human cell. Here, we suggest to take advantage of this fact, by developing inhibitors against one of the bacterial copper transporter: CusCBA. The adaptor protein within this transporter, CusB, plays a critical role in the opening of the whole transporter. We designed a peptide that interfere with its proper function and assembly, and therefore inhibits the opening of the transporter upon copper stress. This study lays the foundation for designing better and novel antibiotics.

biochemistry↗