Celia Valente Romão

This letter describes the development of cryo-EM in Portugal, highlighting a strategy based on research training and the establishment of a national facility with regional support nodes. Our experience demonstrates how access to European research infrastructures can enable the creation of sustainable national platforms without investment in high-end microscopes.

Flavodiiron proteins (FDPs) are NO and/or O ₂ reductases which contain a di-iron catalytic center. Interestingly, they exhibit different selectivities towards each one of these substrates, despite having the same ligands of the iron ions. Escherichia coli FDP is a selective NO reductase that protects this bacterium against nitric oxide by catalyzing two-electron reduction to the nontoxic N ₂ O. Previously, based on kinetic studies, we explored the possible role of two amino acids located in the di-iron second coordination sphere, Lys53 and Tyr271, in modulation of the substrate selectivity of Entamoeba histolytica FDP, a selective O ₂ reductase. In this work, we replaced the structurally equivalent residues in E. coli FDP, Asp52 and Ser262, by those present in the O ₂ -selective FDP and determined their crystal structures in both oxidized and reduced states. Furthermore, the molecular-substrate tunnels were experimentally identified using krypton pressurization of the crystals. The data obtained corroborated previous molecular-dynamics calculations on this FDP. The side chains of residues in both positions 52 and 262 of E. coli FDP variants and wild type are in the vicinity of the shorter intramolecular tunnel, which is suggested to be the exit route for the reaction products N ₂ O and H ₂ O. The E. coli FDP S262Y variant shows photoreduction of the di-iron center and partial loss of electron density in some of its coordinating ligands after X-ray exposure, and these effects are consistent with increased radiation sensitivity. The kinetic properties of the variants towards NO and O ₂ were not significantly different from the wild type, contrary to what was observed previously for E. histolytica FDP.

Repair of Iron Centers (RIC) proteins are present in several pathogens, including Staphylococcus aureus , contributing to resistance to nitroxidative stress and virulence. In this work, we investigated the molecular mechanisms by which S. aureus RIC facilitates iron mobilization, through structural analyses and functional studies. EPR spectroscopy shows that S. aureus RIC contains an antiferromagnetically coupled di‐iron site, undergoing two sequential monoelectronic transitions with a reduction potential of +185 mV. The protein exhibits a stronger affinity for Fe ³⁺ and releases Fe ²⁺ more readily, suggesting facilitated Fe ²⁺ mobilization. The first crystallographic structure for this protein reveals that the di‐iron center is embedded in a four‐helix bundle domain and is coordinated by histidine residues (H87, H211, H132, H167) and bidentate glutamates (E136, E215). Additionally, we observed that RIC contains one protein tunnel formed by M129 and T170 residues, which are shown to facilitate iron release. In S. aureus , the operon encoding Fe–S biogenesis SUF ( SU l F ur mobilization) system contains a cysteine desulfurase SufS, a persulfide transporter SufU, and a putative Fe–S scaffold complex SufBC₂D similar to that of Escherichia coli . We show that RIC promotes the assembly of a Fe–S cluster in E. coli SufBC₂D. In S. aureus , we observed the interactions between SufU–SufD and SufC–SufD, which provide the first evidence for the actual formation of this scaffold in the pathogen. Notably, RIC directly interacts with central components of the Fe–S cluster biogenesis, specifically SufS, SufU, and SufD.

Clostridioides difficile CD25890 is a YicC-like endoribonuclease involved in regulating sporulation initiation, a process critical for the host–host transmission of this anaerobic pathogen. Using comparative transcriptomics we identified a small RNA, SQ528, that accumulates at higher levels in a CD25890 deletion mutant and we show that purified CD25890 cleaves SQ528 in a metal-dependent manner. Moreover, the overexpression of SQ528 increases sporulation under certain nutritional conditions phenocopying a CD25890 deletion mutant. CD25890 is an hexamer in solution and in vivo. An N-terminal domain, which self-interacts as assessed by size exclusion chromatography and a two hybrid assay, is essential for oligomerization of CD25890. A C-terminal domain harbours residues H230, E254, and E258, conserved among orthologues, important for catalysis. AlphaFold2 modelling and cryo-EM suggest an elongated barrel-like structure with an internal cavity lined with basic residues that may aid in RNA binding. We show that CD25890 forms a complex with polynucleotide phosphorylase which combines the endoribonuclease activity of the first with the exonucleolytic activity of the latter and leads to the complete degradation of SQ528. This study identifies a native substrate for the YicC-family of ribonucleases and advances our understanding of the role of CD25890 in sporulation initiation in C. difficile.

Nitroxoline is a bacteriostatic quinoline antibiotic, known to form complexes with metals. Its clinical indications are limited to uncomplicated urinary tract infections, with a susceptibility breakpoint only available for Escherichia coli. Here, we test > 1000 clinical isolates and demonstrate a much broader activity spectrum and species-specific bactericidal activity, including Gram-negative bacteria for which therapeutic options are limited due to multidrug resistance. By combining genetic and proteomic approaches with direct measurement of intracellular metals, we show that nitroxoline acts as a metallophore, inducing copper and zinc intoxication in bacterial cells. The compound displays additional effects on bacterial physiology, including alteration of outer membrane integrity, which underpins nitroxoline’s synergies with large-scaffold antibiotics and resensitization of colistin-resistant Enterobacteriaceae in vitro and in vivo. Furthermore, we identify conserved resistance mechanisms across bacterial species, often leading to nitroxoline efflux.

Non‐structural protein 1 (Nsp1) is a key component of the infectious process caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV2), responsible for the COVID‐19 pandemic. Our previous data demonstrated that Nsp1 can degrade both RNA and DNA in the absence of the ribosome, a process dependent on the metal ions Mn²⁺, Ca²⁺, or Mg²⁺ (Salgueiro et al., SARS‐CoV2 Nsp1 is a metal‐dependent DNA and RNA endonuclease. Biometals. 2024;37:1127–1146). The protein is composed of two structural domains: the N‐terminal domain (NTD) and C‐terminal domain (CTD), connected by a loop. To elucidate the function of each structural domain, we generated four truncated versions of Nsp1 containing either the NTD or the CTD. Our results indicate that the Nsp1^SARS‐CoV2 domains play distinct functional roles. Specifically, the NTD is involved in nucleotide binding and regulation, while the CTD acts as the catalytic domain. Moreover, a tyrosyl radical was detected during the nuclease activity, and an in‐depth analysis of the different constructs suggested that Y136 could be involved in this process. Indeed, our results show that Y136F Nsp1 variant lacks DNA nuclease activity but retains its RNA nuclease activity. Furthermore, we observed that the CTD has a propensity to associate with hydrophobic environments, suggesting that it might associate with cell membranes. However, the cellular function of this association requires further investigation. This study sheds light on the functions of the individual domains of Nsp1, providing valuable insights into its mechanism of action in Coronaviruses.

Nitroxoline is a bacteriostatic quinoline antibiotic, considered a metal chelator inhibiting the activity of RNA-polymerase ¹ . Its clinical indications are limited to uncomplicated urinary tract infections (UTIs), with a clinical susceptibility breakpoint only available for Escherichia coli ² . By testing > 1,000 clinical isolates, here we demonstrate a much broader activity spectrum and species-specific bactericidal activity, including multidrug-resistant Gram-negative bacteria for which therapeutic options are limited due to resistance. By combining systematic genetic and proteomic approaches with direct measurement of intracellular metals, we dissect nitroxoline perturbation of metal homeostasis and unveil additional effects on bacterial physiology. We show that nitroxoline affects outer membrane integrity, synergizing with large-scaffold antibiotics and resensitizing colistin-resistant Enterobacteriaceae in vitro and in vivo . We further characterise resistance mechanisms across E. coli , Acinetobacter baumannii and Klebsiella pneumoniae , recapitulating known E. coli resistance determinants and uncovering novel and conserved mechanisms across species, demonstrating their common effect on nitroxoline efflux.

Introduction to the 5th Biometals WebinarsWelcome to the 5th international webinars series For those who don't already know, these webinars have been set up by the international Biometals society and the journal Biometals with the help of Cassyni platform. • The aim is to promote research in the field of metal interactions in biology and to encourage the interdisciplinary exchange of information at international level. Manganese (Mn) in the spotlight today! Mn, just before iron in the periodic table, is importance is quite often under evaluated compare to iron! As iron Mn is essential but may be toxic due to mainly its redox properties. Mn is a key cofactor in many enzymes and vital system such as photosynthesis… where the tetranuclear manganese cofactor in photosystem 2 catalyzes the key reaction of oxygen production….but The Mn Superoxide dismutase is also a key actor…and many other enzymes contain Mn as cofactor prokaryotic, eukaryotes…and viral!!!! Intracellular or excreted welcome two speakers: John Helmann from Cornell University will be talking about Manganese homeostasis in his favorite playground Bacillus subtilis The 2nd speaker, Celia Romão will be talking about Manganese in a metal‑dependent endonuclease, not just any enzyme, but the one encoded by nsp1, one of the 16 sarsCov2 genes!Managing Manganese in Bacillus subtilisBacteria must adapt to both metal ion limitation and excess. In the model Gram-positive bacterium Bacillus subtilis, the MntR (manganese transport regulator) protein binds Mn(II), represses uptake genes, and activates two genes (mneP, mneS) encoding cation diffusion facilitator proteins that export Mn(II). Genetic studies of mutants dysregulated for Mn homeostasis (mntR) or lacking the major Mn efflux pumps (mneP mneS) revealed a class of mutations with increased expression or activity of two TerC family membrane proteins, MeeF(YceF) and MeeY(YkoY). MeeF and MeeY (metalation of exoenzymes) are Mn efflux proteins that function to support metalation of enzymes that function outside the cell membrane. Unlike MneP and MneS, the MeeF and MeeY proteins do not play a major role in resistance to elevated Mn, although mutations can increase their efflux activity. FY mutant strains, with deletions of both meeF and meeY, are slow growing due to defects in protein secretion and a reduced ability to metalate a key enzyme for cell wall synthesis, the Mn-dependent lipoteichoic acid synthase (LtaS). Consistent with a role in the metalation of Mn-requiring exoenzymes, the MeeY protein is regulated by a Mn-responsive riboswitch. We propose that MeeF and MeeY function, in part, in the co-translocational metalation of secreted enzymes. These findings highlight the challenge of properly metalating enzymes that function outside the cell and may prove relevant to studies of TerC homologs in plants and animals.SARS‑CoV2 Nsp1 is a metal‑dependent DNA and RNA endonucleaseOver recent years, we have been living under a pandemic, caused by the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). One of the major virulence factors of Coronaviruses is the Non-structural protein 1 (Nsp1), known to suppress the host cells protein translation machinery, allowing the virus to produce its own proteins, propagate and invade new cells. To unveil the molecular mechanisms of SARS-CoV2 Nsp1, we have addressed its biochemical and biophysical properties in the presence of calcium, magnesium and manganese. Our findings indicate that the protein in solution is a monomer and binds to both manganese and calcium, with high affinity. Surprisingly, our results show that SARS-CoV2 Nsp1 alone displays metal-dependent endonucleolytic activity towards both RNA and DNA, regardless of the presence of host ribosome. These results show Nsp1 as new nuclease within the coronavirus family. Furthermore, the Nsp1 double variant R124A/K125A presents no nuclease activity for RNA, although it retains activity for DNA, suggesting distinct binding sites for DNA and RNA. Thus, we present for the first time, evidence that the activities of Nsp1 are modulated by the presence of different metals, which are proposed to play an important role during viral infection. This research contributes significantly to our understanding of the mechanisms of action of Coronaviruses.

Over recent years, we have been living under a pandemic, caused by the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). One of the major virulence factors of Coronaviruses is the Non-structural protein 1 (Nsp1), known to suppress the host cells protein translation machinery, allowing the virus to produce its own proteins, propagate and invade new cells. To unveil the molecular mechanisms of SARS-CoV2 Nsp1, we have addressed its biochemical and biophysical properties in the presence of calcium, magnesium and manganese. Our findings indicate that the protein in solution is a monomer and binds to both manganese and calcium, with high affinity. Surprisingly, our results show that SARS-CoV2 Nsp1 alone displays metal-dependent endonucleolytic activity towards both RNA and DNA, regardless of the presence of host ribosome. These results show Nsp1 as new nuclease within the coronavirus family. Furthermore, the Nsp1 double variant R124A/K125A presents no nuclease activity for RNA, although it retains activity for DNA, suggesting distinct binding sites for DNA and RNA. Thus, we present for the first time, evidence that the activities of Nsp1 are modulated by the presence of different metals, which are proposed to play an important role during viral infection. This research contributes significantly to our understanding of the mechanisms of action of Coronaviruses.