The pivotal role of MBD4-ATP7B in the human Cu(i) excretion path as revealed by EPR experiments and all-atom simulations

Copper's essentiality and toxicity require a meticulous mechanism for its acquisition, cellular distribution and excretion, which remains hitherto elusive. Herein, we jointly employed electron paramagnetic resonance spectroscopy and all-atom simulations to resolve the copper trafficking mechanism in humans considering the route travelled by Cu(i) from the metallochaperone Atox1 to the metal binding domains 3 and 4 of ATP7B. Our study shows that Cu(i) in the final part of its extraction pathway is most likely mediated by binding of Atox1 monomer to MBD4 of ATP7B. This interaction takes place through weak metal-stabilized protein-protein interactions

The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

Electron paramagnetic resonance (EPR) spectroscopy has emerged as an ideal biophysical tool to study complex biological processes. EPR spectroscopy can follow minor conformational changes in various proteins as a function of ligand or protein binding or interactions with high resolution and sensitivity. Resolving cellular mechanisms, involving small ligand binding or metal ion transfer, is not trivial and cannot be studied using conventional biophysical tools. In recent years, our group has been using EPR spectroscopy to study the mechanism underlying copper ion transfer in eukaryotic and prokaryotic systems. This mini-review focuses on our achievements following copper metal coordination in the diamagnetic oxidation state, Cu(I), between biomolecules. We discuss the conformational changes induced in proteins upon Cu(I) binding, as well as the conformational changes induced in two proteins involved in Cu(I) transfer. We also consider how EPR spectroscopy, together with other biophysical and computational tools, can identify the Cu(I)-binding sites. This work describes the advantages of EPR spectroscopy for studying biological processes that involve small ligand binding and transfer between intracellular proteins

Advances in Understanding of the Copper Homeostasis in <i>Pseudomonas aeruginosa</i>

Thirty-five thousand people die as a result of more than 2.8 million antibiotic-resistant infections in the United States of America per year. Pseudomonas aeruginosa (P. aeruginosa) is classified a serious threat, the second-highest threat category of the U.S. Department of Health and Human Services. Among others, the World Health Organization (WHO) encourages the discovery and development of novel antibiotic classes with new targets and mechanisms of action without cross-resistance to existing classes. To find potential new target sites in pathogenic bacteria, such as P. aeruginosa, it is inevitable to fully understand the molecular mechanism of homeostasis, metabolism, regulation, growth, and resistances thereof. P. aeruginosa maintains a sophisticated copper defense cascade comprising three stages, resembling those of public safety organizations. These stages include copper scavenging, first responder, and second responder. Similar mechanisms are found in numerous pathogens. Here we compare the copper-dependent transcription regulators cueR and copRS of Escherichia coli (E. coli) and P. aeruginosa. Further, phylogenetic analysis and structural modelling of mexPQ-opmE reveal that this efflux pump is unlikely to be involved in the copper export of P. aeruginosa. Altogether, we present current understandings of the copper homeostasis in P. aeruginosa and potential new target sites for antimicrobial agents or a combinatorial drug regimen in the fight against multidrug resistant pathogens

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 (Met) 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, our outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake

Nitrogen Structure Determination in Treated Fancy Diamonds via EPR Spectroscopy

Color induction in nitrogen-contaminated diamonds was carried out via various procedures that involve irradiation, thermal treatments (annealing), and more. These treatments affect vacancy defect production and atom orientation centers in the diamond lattice. Natural diamonds underwent color enhancement treatments in order to produce green, blue, and yellow fancy diamonds. The aim of this study was to follow the changes occurring during the treatment, mainly by EPR spectroscopy, which is the main source for the determination of the effect of paramagnetic centers (carbon-centered radicals) on the color centers produced via the treatments, but also via visual assessment, fluorescence, UV-vis, and FTIR spectroscopy. The results indicate that diamonds containing high levels of nitrogen contamination are associated with high carbon-centered radical concentrations. Four paramagnetic center structures (N1, N4, and P2/W21) were generated by the treatment. It is suggested that the N4 structure correlates with the formation of blue color centers, whereas yellow color centers are attributed to the presence of N1 species. While to produce blue and yellow colors, a thermal treatment is needed after irradiation, for treated green diamonds, no thermal treatment is needed (only irradiation)

Effect of Diamond Polishing and Thermal Treatment on Carbon Paramagnetic Centers&rsquo; Nature and Structure

Diamonds contain carbon paramagnetic centers (stable carbon radicals) in small concentrations (at the level of ~1 &times; 1012 spins/mg) that can help in elucidating the structure of the nitrogen atoms&rsquo; contaminants in the diamond crystal. All diamonds that undergo polishing are exposed to high temperatures, owing to the friction force during the polishing process, which may affect the carbon-centered radicals&rsquo; concentration and structure. The temperature is increased appreciably; consequently, the black body radiation in the visible range turns orange. During polishing, diamonds emit an orange light (at a wavelength of about 600 nm) that is typical of a black body temperature of 900 &deg;C or higher. Other processes in which color-enhanced diamonds are exposed to high temperatures are thermal treatments or the high-pressure, high-temperature (HPHT) process in which the brown color (resulting from plastic deformation) is bleached. The aim of the study was to examine how thermal treatment and polishing influence the paramagnetic centers in the diamond. For this purpose, four rough diamonds were studied: two underwent a polishing process, and the other two were thermally treated at 650 &deg;C and 1000 &deg;C. The diamonds were analyzed pre- and post-treatment by EPR (Electron Paramagnetic resonance), FTIR (Fourier transform infrared, fluorescence, and their visual appearance. The results indicate that the polishing process results in much more than just thermal heating the paramagnetic centers

Nitrogen Structure Determination in Treated Fancy Diamonds via EPR Spectroscopy

Color induction in nitrogen-contaminated diamonds was carried out via various procedures that involve irradiation, thermal treatments (annealing), and more. These treatments affect vacancy defect production and atom orientation centers in the diamond lattice. Natural diamonds underwent color enhancement treatments in order to produce green, blue, and yellow fancy diamonds. The aim of this study was to follow the changes occurring during the treatment, mainly by EPR spectroscopy, which is the main source for the determination of the effect of paramagnetic centers (carbon-centered radicals) on the color centers produced via the treatments, but also via visual assessment, fluorescence, UV-vis, and FTIR spectroscopy. The results indicate that diamonds containing high levels of nitrogen contamination are associated with high carbon-centered radical concentrations. Four paramagnetic center structures (N1, N4, and P2/W21) were generated by the treatment. It is suggested that the N4 structure correlates with the formation of blue color centers, whereas yellow color centers are attributed to the presence of N1 species. While to produce blue and yellow colors, a thermal treatment is needed after irradiation, for treated green diamonds, no thermal treatment is needed (only irradiation)

EPR Spectroscopy Shows that the Blood Carrier Protein, Human Serum Albumin, Closely Interacts with the N‑Terminal Domain of the Copper Transporter, Ctr1

Copper is an essential metal whose localization within the cells must be carefully controlled to avoid copper dependent redox cycling. Although most of the key proteins involved in cellular copper transfer have been identified, fundamental questions regarding the copper transfer mechanism have yet to be resolved. One of the blood carrier proteins believed to be involved in copper transfer to the cell is human serum albumin (HSA). However, direct evidence for close interaction between HSA and the extracellular domain of the copper transporter Ctr1 has not yet been found. By utilizing EPR spectroscopy, we show here that HSA closely interacts with the first 14 amino acids of the Ctr1, even without the presence of copper ions