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1. Arsenic Exposure Causes Global Changes in the Metalloproteome of Escherichia coli

   https://doi.org/10.3390/microorganisms11020382  

   Larson, James; Tokmina-Lukaszewska, Monika; Fausset, Hunter; Spurzem, Scott; Cox, Savannah; Cooper, Gwendolyn; Copié, Valérie; Bothner, Brian (February 2023, Microorganisms)

   Arsenic is a toxic metalloid with differential biological effects, depending on speciation and concentration. Trivalent arsenic (arsenite, AsIII) is more toxic at lower concentrations than the pentavalent form (arsenate, AsV). In E. coli, the proteins encoded by the arsRBC operon are the major arsenic detoxification mechanism. Our previous transcriptional analyses indicate broad changes in metal uptake and regulation upon arsenic exposure. Currently, it is not known how arsenic exposure impacts the cellular distribution of other metals. This study examines the metalloproteome of E. coli strains with and without the arsRBC operon in response to sublethal doses of AsIII and AsV. Size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICPMS) was used to investigate the distribution of five metals (56Fe, 24Mg, 66Zn, 75As, and 63Cu) in proteins and protein complexes under native conditions. Parallel analysis by SEC-UV-Vis spectroscopy monitored the presence of protein cofactors. Together, these data reveal global changes in the metalloproteome, proteome, protein cofactors, and soluble intracellular metal pools in response to arsenic stress in E. coli. This work brings to light one outcome of metal exposure and suggests that metal toxicity on the cellular level arises from direct and indirect effects. 

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2. Protein-Cadmium Interactions in Crowded Biomolecular Environments Probed by In-cell and Lysate NMR Spectroscopy

   Sachin S. Katti, Tatyana I. (November 2023, bioRxiv)

   One of the mechanisms by which toxic metal ions interfere with cellular functions is ionic mimicry, where they bind to protein sites in lieu of native metals Ca2+ and Zn2+. The influence of crowded intracellular environments on these interactions is not well understood. Here, we demonstrate the application of in-cell and lysate NMR spectroscopy to obtain atomic-level information on how a potent environmental toxin cadmium interacts with its protein targets. The experiments, conducted in intact E. coli cells and their lysates, revealed that Cd2+ can profoundly affect the quinary interactions of its protein partners, and can replace Zn2+ in both labile and non-labile protein structural sites without significant perturbation of the membrane binding function. Surprisingly, in crowded molecular environments Cd2+ can effectively target not only all-sulfur and mixed sulfur/nitrogen but also all-oxygen coordination sites. The sulfur-rich coordination environments show significant promise for bioremedial applications, as demonstrated by the ability of the designed protein scaffold α3DIV to sequester intracellular cadmium. Our data suggests that in-cell NMR spectroscopy is a powerful tool for probing interactions of toxic metal ions with their potential protein targets, and for the assessment of potency of sequestering agents. 

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3. Structure and ion-release mechanism of PIB-4-type ATPases

   https://doi.org/10.7554/eLife.73124  

   Grønberg, Christina; Hu, Qiaoxia; Mahato, Dhani Ram; Longhin, Elena; Salustros, Nina; Duelli, Annette; Lyu, Pin; Bågenholm, Viktoria; Eriksson, Jonas; Rao, Komal Umashankar; et al (December 2021, eLife)

   Heavy metals such as zinc and cobalt are toxic at high levels, yet most organisms need tiny amounts for their cells to work properly. As a result, proteins studded through the cell membrane act as gatekeepers to finetune import and export. These proteins are central to health and disease; their defect can lead to fatal illnesses in humans, and they also help bacteria infect other organisms. Despite their importance, little is known about some of these metal-export proteins. This is particularly the case for P IB-4 -ATPases, a subclass found in plants and bacteria and which includes, for example, a metal transporter required for bacteria to cause tuberculosis. Intricate knowledge of the three-dimensional structure of these proteins would help to understand how they select metals, shuttle the compounds in and out of cells, and are controlled by other cellular processes. To reveal this three-dimensional organisation, Grønberg et al. used X-ray diffraction, where high-energy radiation is passed through crystals of protein to reveal the positions of atoms. They focused on a type of PIB-4-ATPases found in bacteria as an example. The work showed that the protein does not contain the metal-binding regions seen in other classes of metal exporters; however, it sports unique features that are crucial for metal transport such as an adapted pathway for the transport of zinc and cobalt across the membrane. In addition, Grønberg et al. tested thousands of compounds to see if they could block the activity of the protein, identifying two that could kill bacteria. This better understanding of how PIB-4-ATPases work could help to engineer plants capable of removing heavy metals from contaminated soils, as well as uncover new compounds to be used as antibiotics. 

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4. Thermodynamic origin of the affinity, selectivity, and domain specificity of metallothionein for essential and toxic metal ions

   https://doi.org/10.1093/mtomcs/mfae041  

   Quinn, Colette_F; Wilcox, Dean_E (September 2024, Metallomics)

   Abstract The small Cys-rich protein metallothionein (MT) binds several metal ions in clusters within two domains. While the affinity of MT for both toxic and essential metals has been well studied, the thermodynamics of this binding has not. We have used isothermal titration calorimetry measurements to quantify the change in enthalpy (ΔH) and change in entropy (ΔS) when metal ions bind to the two ubiquitous isoforms of MT. The seven Zn2+ that bind sequentially at pH 7.4 do so in two populations with different coordination thermodynamics, an initial four that bind randomly with individual tetra-thiolate coordination and a subsequent three that bind with bridging thiolate coordination to assemble the metal clusters. The high affinity of MT for both populations is due to a very favourable binding entropy that far outweighs an unfavourable binding enthalpy. This originates from a net enthalpic penalty for Zn2+ displacement of protons from the Cys thiols and a favourable entropic contribution from the displaced protons. The thermodynamics of other metal ions binding to MT were determined by their displacement of Zn2+ from Zn7MT and subtraction of the Zn2+-binding thermodynamics. Toxic Cd2+, Pb2+, and Ag+, and essential Cu+, also bind to MT with a very favourable binding entropy but a net binding enthalpy that becomes increasingly favourable as the metal ion becomes a softer Lewis acid. These thermodynamics are the origin of the high affinity, selectivity, and domain specificity of MT for these metal ions and the molecular basis for their in vivo binding competition. 

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5. mebipred : identifying metal-binding potential in protein sequence

   https://doi.org/10.1093/bioinformatics/btac358  

   Aptekmann, A. A.; Buongiorno, J.; Giovannelli, D.; Glamoclija, M.; Ferreiro, D. U.; Bromberg, Y.; Cowen, ed., Lenore (May 2022, Bioinformatics)

   Abstract Motivationmetal-binding proteins have a central role in maintaining life processes. Nearly one-third of known protein structures contain metal ions that are used for a variety of needs, such as catalysis, DNA/RNA binding, protein structure stability, etc. Identifying metal-binding proteins is thus crucial for understanding the mechanisms of cellular activity. However, experimental annotation of protein metal-binding potential is severely lacking, while computational techniques are often imprecise and of limited applicability. Resultswe developed a novel machine learning-based method, mebipred, for identifying metal-binding proteins from sequence-derived features. This method is over 80% accurate in recognizing proteins that bind metal ion-containing ligands; the specific identity of 11 ubiquitously present metal ions can also be annotated. mebipred is reference-free, i.e. no sequence alignments are involved, and is thus faster than alignment-based methods; it is also more accurate than other sequence-based prediction methods. Additionally, mebipred can identify protein metal-binding capabilities from short sequence stretches, e.g. translated sequencing reads, and, thus, may be useful for the annotation of metal requirements of metagenomic samples. We performed an analysis of available microbiome data and found that ocean, hot spring sediments and soil microbiomes use a more diverse set of metals than human host-related ones. For human microbiomes, physiological conditions explain the observed metal preferences. Similarly, subtle changes in ocean sample ion concentration affect the abundance of relevant metal-binding proteins. These results highlight mebipred’s utility in analyzing microbiome metal requirements. Availability and implementationmebipred is available as a web server at services.bromberglab.org/mebipred and as a standalone package at https://pypi.org/project/mymetal/. Supplementary informationSupplementary data are available at Bioinformatics online. 

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