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Abstract: A family of bacterial copper storage proteins (the Csps) possess thiolate-lined four-helix bundles whose cores can be filled with Cu(I) ions. The majority of Csps are cytosolic (Csp3s), and in vitro studies carried out to date indicate that the Csp3s from Methylosinus trichosporium OB3b (MtCsp3), Bacillus subtilis (BsCsp3), and Streptomyces lividans (SlCsp3) are alike. Bioinformatics have highlighted homologues with potentially different Cu(I)-binding properties from these characterized “classical” Csp3s. Determination herein of the crystal structure of the protein (RkCsp3) from the methanotroph Methylocystis sp. strain Rockwell with Cu(I) bound identifies this as the first studied example of a new subgroup of Csp3s. The most significant structural difference from classical Csp3s is the presence of only two Cu(I) sites at the mouth of the bundle via which Cu(I) ions enter and leave. This is due to the absence of three Cys residues and a His-containing motif, which allow classical Csp3s to bind five to six Cu(I) ions in this region. Regardless, RkCsp3 exhibits rapid Cu(I) binding and the fastest measured Cu(I) removal rate for a Csp3 when using high-affinity ligands as surrogate partners. New experiments on classical Csp3s demonstrate that their His-containing motif is not essential for fast Cu(I) uptake and removal. Other structural features that could be important for these functionally relevant in vitro properties are discussed.

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Abstract: Evolution creates functional diversity of proteins, the essential building blocks of all biological systems. However, studies of natural proteins sampled across the tree of life and evaluated in a single experimental system are lacking. Almost half of enzymes require metals, and metalloproteins tend to optimally utilize the physicochemical properties of a specific metal co-factor. Life must adapt to changes in metal bioavailability, including those during the transition from anoxic to oxic Earth or pathogens’ exposure to nutritional immunity. These changes can challenge the ability of metalloenzymes to maintain activity, presumptively driving their evolution. Here we studied metal-preference evolution within the natural diversity of the iron/manganese superoxide dismutase (SodFM) family of reactive oxygen species scavengers. We identified and experimentally verified residues with conserved roles in determining metal preference that, when combined with an understanding of the protein’s evolutionary history, improved prediction of metal utilization across the five SodFM subfamilies defined herein. By combining phylogenetics, biochemistry and structural biology, we demonstrate that SodFM metal utilization can be evolutionarily fine tuned by sliding along a scale between perfect manganese and iron specificities. Over the history of life, SodFM metal preference has been modulated multiple independent times within different evolutionary and ecological contexts, and can be changed within short evolutionary timeframes.

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Abstract: SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp’s, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 μM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.

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Abstract: Operando soft and hard X-ray spectroscopic techniques were used in combination with plane-wave density functional theory (DFT) simulations to rationalize the enhanced activities of Zn-containing Cu nanostructured electrocatalysts in the electrocatalytic CO2 hydrogenation reaction. We show that at a potential for CO2 hydrogenation, Zn is alloyed with Cu in the bulk of the nanoparticles with no metallic Zn segregated; at the interface, low reducible Cu(I)–O species are consumed. Additional spectroscopic features are observed, which are identified as various surface Cu(I) ligated species; these respond to the potential, revealing characteristic interfacial dynamics. Similar behavior was observed for the Fe–Cu system in its active state, confirming the general validity of this mechanism; however, the performance of this system deteriorates after successive applied cathodic potentials, as the hydrogen evolution reaction then becomes the main reaction pathway. In contrast to an active system, Cu(I)–O is now consumed at cathodic potentials and not reversibly reformed when the voltage is allowed to equilibrate at the open-circuit voltage; rather, only the oxidation to Cu(II) is observed. We show that the Cu–Zn system represents the optimal active ensembles with stabilized Cu(I)–O; DFT simulations rationalize this observation by indicating that Cu–Zn–O neighboring atoms are able to activate CO2, whereas Cu–Cu sites provide the supply of H atoms for the hydrogenation reaction. Our results demonstrate an electronic effect exerted by the heterometal, which depends on its intimate distribution within the Cu phase and confirms the general validity of these mechanistic insights for future electrocatalyst design strategies.

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Abstract: Bioinspired de novo self-assembling peptides have been widely used for the development of soft biomaterials for a wide variety of biomedical and pharmaceutical applications, such as cell scaffolding for tissue engineering1, controlled and localised drug delivery2, biosensing3, and many others. The meticulous control of peptide-based nanomaterial properties over the length scale, by molecular design, remains the main challenge for tailoring biomaterials properties to meet the application needs. In our group, we have recently adopted a minimalistic molecular engineering approach for the development of Ultrashort Ionic-complementary Constrained Peptides (UICPs), which were rationally designed to self-assemble into amphiphilic β-sheet nanofibers with unique hydrogelation properties and surface activity.4 We have previously demonstrated the crucial role played by aromatic stacking for the formation and thermodynamic stabilisation of UICP β-sheet structures. Herein, we will show how charge interactions can be manipulated for fine tuning molecular self-assembly, morphology and size of nanofibrous structures formation and viscoelasticity of UICP hydrogels. A library of 18 peptide sequences (4-5 residues long) was developed to study the effect of the sequence net charge, charge density distribution, reversal of charge order and ionic self-complementarity on their propensity towards self-assembly and gelation. Interestingly, 12 of these peptides self-assembled into β-sheet nanofibrous structures forming hydrogels at pH 4.5-5, as confirmed by ATR-FTIR, SEM, TEM, SAXS and oscillatory rheology. Full control over β-sheet content (ranging from ~30-80%), fibre morphology (thin fibrils, thick straight fibre bundles, twisted helical nanofibres, flat nanoribbons and nanotubes) and sizes (~4-67 nm in diameter), as well as gelation (critical gelation concentrations ranging from <7.5 to >100 mM) and viscoelastic properties (storage moduli G’ ~0.1-100 KPa) was achieved by the careful positioning of both Glu and Lys residues at both C- and N-termini, in the sequence core and on both the hydrophilic and hydrophobic faces of the peptide chain. In essence, this design approach enabled/disabled lateral growth along the β-sheet ladder via electrostatic attraction (counter charge, anion-pi and cation-pi)/repulsion, hence controlling fibre thickness, morphology, entanglement, and the resulting viscoelasticity of the system. Our UICPs platform thus provides the flexibility in peptide molecular design for the manufacturing of soft biomaterials with versatile properties that can be in future tailored to the relevant biomedical application.