Biology subjects

Ruthstein, S.

Publications and source records attributed to Ruthstein, S..

6 recordsLinked to original sources

bioRxiv2026

CopY as a Transcriptional Duet Orchestrating Copper Homeostasis via Asymmetry-Dependent DNA Affinity

Copper is essential yet potentially toxic, requiring tight regulation of intracellular concentration. The copper-responsive transcriptional repressor CopY is unusual among bacterial metalloregulators because it occupies two operator sites within the cop promoter. Although two CopY dimers bind these operators independently, the mechanistic significance of this dual-operator architecture remains unclear. Here, we combine biochemical assays, circular dichroism (CD), and electron paramagnetic resonance (EPR) distance measurements to define the molecular basis of CopY-mediated repression. CopY binds the proximal operator with higher affinity than the distal operator. CD measurements further show that DNA binding increases CopY thermal stability, with greater stabilization by the proximal operator. Conversely, DNA-based EPR measurements reveal comparable CopY-induced compaction of both operators. Thus, protein stabilization and DNA deformation represent distinct components of repression. Moreover, site-directed spin labeling and EPR demonstrate that the metal binding domain is conformationally heterogeneous and highly flexible. DNA binding has only a marginal effect on this flexibility. Thus, the metal site is not a rigid pocket but a dynamic one, even in the DNA-bound repressor, a property that may facilitate Cu(I) capture and hence derepression once copper levels rise. We propose that the proximal operator serves as a high-affinity anchoring site, whereas the distal operator primarily promotes promoter remodeling, and that the plasticity of the metal binding domain is retained in the DNA-bound repressor. Together, these findings support a "transcriptional duet" model in which two CopY dimers bound to non-equivalent operators make distinct and complementary contributions to repression.

biophysics↗

bioRxiv2026

A bacterial extracellular matrix protein forms a supramolecular metallogel

The microbial extracellular matrix (ECM) is a complex network of self-secreted biopolymers uniting the cells in biofilms, providing them with structural integrity, and contributing to their elevated resistance to antibiotic treatments. Recently, there is a growing realization that a regulated, bidirectional cross-talk of bacteria and ECM confers biofilms with tissue-like traits, however, the mechanisms of spatio-temporal self-organisation of ECM and its regulation are still poorly understood. In the model organism for biofilm formation Bacillus subtilis, TasA is the major protein component of the extracellular matrix. We recently showed that TasA, isolated in the form of stable and structured globules, assembles into elongated and ordered fibers via a donor-strand complementation mechanism. In this study, we discovered that in the presence of zinc metal ions, TasA is able to form hydrogels with > 97% water content. Electron- and atomic force-microscopies as well as small angle X-ray scattering measurements show that cross-linking with zinc ions induces a transition in TasA morphology from one-dimensional fibers to two-dimensional sheets. Electron paramagnetic resonance measurements then show that such a significant morphological shift is associated with molecular changes in the coordination environment of zinc ions, which lead to structural changes at the protein level. When assembling into macroscopic networks, TasA-Zn metallogels exhibit viscoelastic properties and a fast recovery following an excessive strain. These metallogels represent a novel class of bacterially-derived ECMs that form easily at room temperature without covalent crosslinking, and may be used as a natural matrix-mimics in biofilm models for infection studies.

biochemistry↗

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↗

bioRxiv2021

The Conformational Plasticity of the Selectivity Filter Methionines Controls the In-Cell Cu(I) Uptake through the CTR1 transporter

Copper is a trace element vital to many cellular functions. Yet its abnormal levels are toxic to cells, provoking a variety of severe diseases. The high affinity Copper Transporter 1 (CTR1), being the main in-cell copper (Cu(I)) entry route, tightly regulates its cellular uptake via a still elusive mechanism. Here, all-atoms simulations unlock the molecular terms of Cu(I) transport in eukaryotes disclosing that the two Methionine triads, forming the selectivity filter, play an unprecedented dual role both enabling selective Cu(I) transport and regulating its uptake-rate thanks to an intimate coupling between the conformational plasticity of their bulky side chains and the number of bound Cu(I) ions. Namely, the Met residues act as a gate reducing the Cu(I) import-rate when two ions simultaneously bind to CTR1. This may represent an elegant autoregulatory mechanism through which CTR1 protects the cells from excessively high, and hence toxic, in-cell Cu(I) levels. Overall, these outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/467269v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@75fbe6org.highwire.dtl.DTLVardef@1ece24eorg.highwire.dtl.DTLVardef@fda950org.highwire.dtl.DTLVardef@1770038_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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↗

bioRxiv2019

Exploring the role of the various methionine residues in the Escherichia coli CusB adapter protein

The dissemination of resistant pathogenic microbes has become one of the most challenging problems that modern medicine has faced. Developing novel drugs based on new molecular targets that previously were not targeted, is therefore the highest priority in antibiotics research. One approach that has been recently suggested is to inhibit copper transporters in prokaryotic systems. Copper is required for many biological pathways, but sometimes it can harm the cell. Pathogenic systems have a highly sophisticated copper-regulation network; therefore, a better understanding of how this network operates at the molecular level should assist in developing the next generation of antibiotics. The CusB protein is part of the CusCBA periplasmic Cu(I) efflux system in Gram-negative bacteria, and it was recently reported to play a key role in the functioning of the whole CusCBA system, in which conformational changes as well as the assembly/disassembly process control the opening of the transporter. More knowledge of the underlying mechanism is needed to attain a full understanding of CusB functioning, which is associated with targeting specific and crucial residues in CusB. Here, we combine in-vitro structural measurements, which used EPR spectroscopy and UV-Vis measurements, with cell experiments to explore the role of the various methionine residues in CusB. We targeted two methionine residues (M227 and M241) that are essential for the proper function of CusB.

biophysics↗