Small molecule MarR modulators potentiate metronidazole antibiotic activity in aerobic E. coli by inducing activation by the nitroreductase NfsA.

Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.

Integrated Experimental and Theoretical Investigation of Copper Active Site Properties of a Lytic Polysaccharide Monooxygenase from Serratia marcescens.

In this paper, we employed a multidisciplinary approach, combining experimental techniques and density functional theory (DFT) calculations to elucidate key features of the copper coordination environment of the bacterial lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens (SmAA10). The structure of the holo-enzyme was successfully obtained by X-ray crystallography. We then determined the copper(II) binding affinity using competing ligands and observed that the affinity of the histidine brace ligands for copper is significantly higher than previously described. UV-vis, advanced electron paramagnetic resonance (EPR), and X-ray absorption spectroscopy (XAS) techniques, including high-energy resolution fluorescence detected (HERFD) XAS, were further used to gain insight into the copper environment in both the Cu(II) and Cu(I) redox states. The experimental data were successfully rationalized by DFT models, offering valuable information on the electronic structure and coordination geometry of the copper center. Finally, the Cu(II)/Cu(I) redox potential was determined using two different methods at ca. 350 mV vs NHE and rationalized by DFT calculations. This integrated approach not only advances our knowledge of the active site properties of SmAA10 but also establishes a robust framework for future studies of similar enzymatic systems.

Large dynamics of a phase separating arginine-glycine-rich domain revealed via nuclear and electron spins.

Liquid-liquid phase separation is the key process underlying formation of membrane-less compartments in cells. A highly dynamic cellular body with rapid component exchange is Cajal body (CB), which supports the extensive compositional dynamics of the RNA splicing machinery, spliceosome. Here, we select an arginine-glycine (RG)-rich segment of coilin, the major component of CB, establish its RNA-induced phase separation, and through combined use of nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) probes, interrogate its dynamics within the crowded interior of formed droplets. Taking advantage of glycine-based singlet-states, we show that glycines retain a large level of sub-nanoseconds dynamics inside the coilin droplets. Furthermore, the continuous-wave (CW) and electron-electron dipolar (PELDOR) and electron-nucleus hyperfine coupling EPR data (HYSCORE) support the RNA-induced formation of dynamic coilin droplets with high coilin peptide concentrations. The combined NMR and EPR data reveal the high dynamics of the RG-rich coilin within droplets and suggest its potential role in the large dynamics of CBs.

Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions.

A copper-dependent self-cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46-nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub-structures that flank a highly conserved catalytic core. The DNA self-cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site-specific cleavage of the substrate are two key-aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the "metal-soup" scenario, also known as "super-stoichiometric" ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids-based enzymes. The focus on the endogenous paramagnetic center (Cu2+) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.

NMR-identification of the interaction between BRCA1 and the intrinsically disordered monomer of the Myc-associated factor X.

The breast cancer susceptibility 1 (BRCA1) protein plays a pivotal role in modulating the transcriptional activity of the vital intrinsically disordered transcription factor MYC. In this regard, mutations of BRCA1 and interruption of its regulatory activity are related to hereditary breast and ovarian cancer (HBOC). Interestingly, so far, MYC's main dimerization partner MAX (MYC-associated factor X) has not been found to bind BRCA1 despite a high sequence similarity between both oncoproteins. Herein, we show that a potential reason for this discrepancy is the heterogeneous conformational space of MAX, which encloses a well-documented folded coiled-coil homodimer as well as a less common intrinsically disordered monomer state-contrary to MYC, which exists mostly as intrinsically disordered protein in the absence of any binding partner. We show that when the intrinsically disordered state of MAX is artificially overpopulated, the binding of MAX to BRCA1 can readily be observed. We characterize this interaction by nuclear magnetic resonance (NMR) spectroscopy chemical shift and relaxation measurements, complemented with ITC and SAXS data. Our results suggest that BRCA1 directly binds the MAX monomer to form a disordered complex. Though probed herein under biomimetic in-vitro conditions, this finding can potentially stimulate new perspectives on the regulatory network around BRCA1 and its involvement in MYC:MAX regulation.

Magnetic resonance investigation of conformational responses of tau protein to specific phosphorylation.

Intrinsically disordered proteins (IDPs) are known to adopt many rapidly interconverting structures, making it difficult to pinpoint the specific conformational states that are relevant for their function. Tau is an important IDP, and its conformation is known to be affected by post-translational modifications (PTMs), such as phosphorylation. To investigate the effect of specific phosphorylation on full-length Tau's dynamic global conformation, we employed a combination of nuclear magnetic resonance-based paramagnetic relaxation interference methods and electron paramagnetic resonance spectroscopy. By reproducing the AT8 epitope, comprising exclusive phosphorylation at residues S202 and T205, we were able to identify conformations specific to phosphorylated Tau, which exhibited a tendency towards less compact states. These mechanistic details are of significance to understand the path leading from soluble Tau to the ordered structure of Tau fibers. This approach proved to be successful for studying the conformational changes of (phosphorylated) full-length Tau and can potentially be extended to the study of other IDPs that undergo various PTMs.

Intrinsic Flexibility beyond the Highly Ordered DNA Tetrahedron: An Integrative Spectroscopic and Molecular Dynamics Approach.

Since the introduction of DNA-based architectures, in the past decade, DNA tetrahedrons have aroused great interest. Applications of such nanostructures require structural control, especially in the perspective of their possible functionalities. In this work, an integrated approach for structural characterization of a tetrahedron structure is proposed with a focus on the fundamental biophysical aspects driving the assembly process. To address such an issue, spin-labeled DNA sequences are chemically synthesized, self-assembled, and then analyzed by Continuous-Wave (CW) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopy. Interspin distance measurements based on PELDOR/DEER techniques combined with molecular dynamics (MD) thus revealed unexpected dynamic heterogeneity and flexibility of the assembled structures. The observation of flexibility in these ordered 3D structures demonstrates the sensitivity of this approach and its effectiveness in accessing the main dynamic and structural features with unprecedented resolution.

Degradation mechanisms of organic compounds in molten hydroxide salts: a radical reaction yielding H2 and graphite.

Molten salts are used in various waste treatments, such as recycling, recovery or making inert. Here, we present a study of the degradation mechanisms of organic compounds in molten hydroxide salts. Molten salt oxidation (MSO) using carbonates, hydroxides and chlorides is known for the treatment of hazardous waste, organic material or metal recovery. This process is described as an oxidation reaction due to the consumption of O2 and formation of H2O and CO2. We have treated various organic products, carboxylic acids, polyethylene and neoprene with molten hydroxides at 400 °C. However, the reaction products obtained in these salts, especially carbon graphite and H2 without CO2 emission, challenges the previous mechanisms described for the MSO process. Combining several analyses of the solid residues and the gas produced during the reaction of organic compounds in molten hydroxides (NaOH-KOH), we demonstrate that these mechanisms are radical-based instead of oxidative. We also show that the obtained end products are highly recoverable graphite and H2, which opens a new way of recycling plastic residues.

Chiral Discrimination Mechanisms by Silylated-Acetylated Cyclodextrins: Superficial Interactions vs. Inclusion.

Cyclodextrin derivatives constitute a powerful class of auxiliary agents for the discrimination of apolar chiral substrates. Both host-guest inclusion phenomena and interactions with the derivatizing groups located on the surface of the macrocycle could drive the enantiodiscrimination; thus, it is important to understand the role that these processes play in the rational design of new chiral selectors. The purpose of this study is to compare via nuclear magnetic resonance (NMR) spectroscopy the efficiency of silylated-acetylated α-, ß-, and γ-cyclodextrins in the chiral discrimination of 1,1,1,3,3-pentafluoro-2-(fluoromethoxy)-3-methoxypropane (compound B) and methyl 2-chloropropionate (MCP). NMR DOSY (Diffusion Ordered SpectroscopY) experiments were conducted for the determination of the bound molar fractions and the association constants, whereas ROESY (Rotating-frame Overhauser Enhancement SpectroscopY) measurements provided information on the hosts' conformation and on the interaction phenomena with the guests. Compound B, endowed with fluorinated moieties, is not deeply included due to attractive Si-F interactions occurring at the external surface of the cyclodextrins. Therefore, a low selectivity toward the size of cyclodextrin cavity is found. By contrast, enantiodiscrimination of MCP relies on the optimal fitting between the size of the guest and that of the cyclodextrin cavity.

Large-Scale Conformational Changes of FhaC Provide Insights Into the Two-Partner Secretion Mechanism.

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane ß barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TpsB transporter FhaC. This revealed substantial, spontaneous conformational changes on a slow time scale, with parts of the POTRA2 domain approaching the lipid bilayer and the protein's surface loops. Specifically, our data indicate that an amphipathic POTRA2 ß hairpin can insert into the ß barrel. We propose that these motions enlarge the channel and initiate substrate secretion. Our data propose a solution to the conundrum how TpsB transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

The 'hidden side' of spin labelled oligonucleotides: Molecular dynamics study focusing on the EPR-silent components of base pairing.

Nitroxide labels are combined with nucleic acid structures and are studied using electron paramagnetic resonance experiments (EPR). As X-ray/NMR structures are unavailable with the nitroxide labels, detailed residue level information, down to atomic resolution, about the effect of these nitroxide labels on local RNA structures is currently lacking. This information is critical to evaluate the choice of spin label. In this study, we compare and contrast the effect of TEMPO-based (NT) and rigid spin (Ç) labels (in both 2'-O methylated and not-methylated forms) on RNA duplexes. We also investigate sequence- dependent effects of NT label on RNA duplex along with the more complex G-quadruplex RNA. Distances measured from molecular dynamics simulations between the two spin labels are in agreement with the EPR experimental data. To understand the effect of labelled oligonucleotides on the structure, we studied the local base pair geometries and global structure in comparison with the unlabelled structures. Based on the structural analysis, we can conclude that TEMPO-based and Ç labels do not significantly perturb the base pair arrangements of the native oligonucleotide. When experimental structures for the spin labelled DNA/RNA molecules are not available, general framework offered by the current study can be used to provide information critical to the choice of spin labels to facilitate future EPR studies.

A Switch between Two Intrinsically Disordered Conformational Ensembles Modulates the Active Site of a Basic-Helix-Loop-Helix Transcription Factor.

We report a conformational switch between two distinct intrinsically disordered subensembles within the active site of a transcription factor. This switch highlights an evolutionary benefit conferred by the high plasticity of intrinsically disordered domains, namely, their potential to dynamically sample a heterogeneous conformational space housing multiple states with tailored properties. We focus on proto-oncogenic basic-helix-loop-helix (bHLH)-type transcription factors, as these play key roles in cell regulation and function. Despite intense research efforts, the understanding of structure-function relations of these transcription factors remains incomplete as they feature intrinsically disordered DNA-interaction domains that are difficult to characterize, theoretically as well as experimentally. Here we characterize the structural dynamics of the intrinsically disordered region DNA-binding site of the vital MYC-associated transcription factor X (MAX). Integrating nuclear magnetic resonance (NMR) measurements, molecular dynamics (MD) simulations, and electron paramagnetic resonance (EPR) measurements, we show that, in the absence of DNA, the binding site of the free MAX2 homodimer samples two intrinsically disordered conformational subensembles. These feature distinct structural properties: one subensemble consists of a set of highly flexible and spatially extended conformers, while the second features a set of "hinged" conformations. In this latter ensemble, the disordered N-terminal tails of MAX2 fold back along the dimer, forming transient long-range contacts with the HLH-region and thereby exposing the DNA binding site to the solvent. The features of these divergent substates suggest two mechanisms by which protein conformational dynamics in MAX2 might modulate DNA-complex formation: by enhanced initial recruitment of free DNA ligands, as a result of the wider conformational space sampled by the extended ensemble, and by direct exposure of the binding site and the corresponding strong electrostatic attractions presented while in the hinged conformations.

A trade-off for covalent and intercalation binding modes: a case study for Copper (II) ions and singly modified DNA nucleoside.

Selective binding to nucleic acids and, more generally, to biopolymers, very often requires at a minimum the presence of specific functionalities and precise spatial arrangement. DNA can fold into defined 3D structures upon binding to metal centers and/or lanthanides. Binding efficiency can be boosted by modified nucleosides incorporated into DNA sequences. In this work the high selectivity of modified nucleosides towards copper (II) ions, when used in the monomeric form, is unexpectedly and drastically reduced upon being covalently attached to the DNA sequence in single-site scenario. Surprisingly, such selectivity is partially retained upon non-covalent (i.e. intercalation) mixture formed by native DNA duplex and a nucleoside in the monomeric form. Exploiting the electron spin properties of such different and rich binding mode scenarios, 1D/2D pulsed EPR experiments have been used and tailored to differentiate among the different modes. An unusual correlation of dispersion of hyperfine couplings and strength of the binding mode(s) is described.

Conformational tuning of a DNA-bound transcription factor.

Transcription factors are involved in many cellular processes that take place remote from their cognate DNA sequences. The efficiencies of these activities are thus in principle counteracted by high binding affinities of the factors to their cognate DNAs. Models such as facilitated diffusion or dissociation address this apparent contradiction. We show that the MYC associated transcription factor X (MAX) undergoes nanoscale conformational fluctuations in the DNA-bound state, which is consistent with facilitated dissociation from or diffusion along DNA strands by transiently reducing binding energies. An integrative approach involving EPR, NMR, crystallographic and molecular dynamics analyses demonstrates that the N-terminal domain of MAX constantly opens and closes around a bound DNA ligand thereby dynamically tuning the binding epitope and the mode of interaction.

Sample Ripening through Nanophase Separation Influences the Performance of Dynamic Nuclear Polarization.

Mixtures of water and glycerol provide popular matrices for low-temperature spectroscopy of vitrified samples. However, they involve counterintuitive physicochemical properties, such as spontaneous nanoscopic phase separations (NPS) in solutions that appear macroscopically homogeneous. We demonstrate that such phenomena can substantially influence the efficiency of dynamic nuclear polarization (DNP) by factors up to 20 % by causing fluctuations in local concentrations of polarization agents (radicals). Thus, a spontaneous NPS of water/glycerol mixtures that takes place on time scales on the order of 30-60 min results in a confinement of polarization agents in nanoscopic water-rich vesicles, which in return affects the DNP. Such effects were found for three common polarization agents, TEMPOL, AMUPol and Trityl.

Unanticipated coordination of tris buffer to the Radical SAM cluster of the RimO methylthiotransferase.

Radical SAM enzymes generally contain a [4Fe-4S](2+/1+) (RS cluster) cluster bound to the protein via the three cysteines of a canonical motif CxxxCxxC. The non-cysteinyl iron is used to coordinate SAM via its amino-carboxylate moiety. The coordination-induced proximity between the cluster acting as an electron donor and the adenosyl-sulfonium bond of SAM allows for the homolytic cleavage of the latter leading to the formation of the reactive 5'-deoxyadenosyl radical used for substrate activation. Most of the structures of Radical SAM enzymes have been obtained in the presence of SAM, and therefore, little is known about the situation when SAM is not present. In this report, we show that RimO, a methylthiotransferase belonging to the radical SAM superfamily, binds a Tris molecule in the absence of SAM leading to specific spectroscopic signatures both in Mössbauer and pulsed EPR spectroscopies. These data provide a cautionary note for researchers who work with coordinative unsaturated iron sulfur clusters.

Fine-tuning of a radical-based reaction by radical S-adenosyl-L-methionine tryptophan lyase.

The radical S-adenosyl-L-methionine tryptophan lyase NosL converts L-tryptophan into 3-methylindolic acid, which is a precursor in the synthesis of the thiopeptide antibiotic nosiheptide. Using electron paramagnetic resonance spectroscopy and multiple L-tryptophan isotopologues, we trapped and characterized radical intermediates that indicate a carboxyl fragment migration mechanism for NosL. This is in contrast to a proposed fragmentation-recombination mechanism that implied Cα-Cß bond cleavage of L-tryptophan. Although NosL resembles related tyrosine lyases, subtle substrate motions in its active site are responsible for a fine-tuned radical chemistry, which selects the Cα-C bond for disruption. This mechanism highlights evolutionary adaptation to structural constraints in proteins as a route to alternative enzyme function.

Orientation selection in distance measurements between nitroxide spin labels at 94 GHz EPR with variable dual frequency irradiation.

Pulsed electron-electron double resonance (PELDOR, also known as DEER) has become a method of choice to measure distances in biomolecules. In this work we show how the performance of the method can be improved at high EPR frequencies (94 GHz) using variable dual frequency irradiation in a dual mode cavity in order to obtain enhanced resolution toward orientation selection. Dipolar evolution traces of a representative RNA duplex and an α-helical peptide were analysed in terms of possible bi-radical structures by considering the inherent ambiguity of symmetry-related solutions.

Synthesis and characterization of RNA containing a rigid and nonperturbing cytidine-derived spin label.

The nitroxide-containing nucleoside Çm is reported as the first rigid spin label for paramagnetic modification of RNA by solid-phase synthesis. The spin label is well accommodated in several RNA secondary structures as judged by its minor effect on the thermodynamic stability of hairpin and duplex RNA. Electron paramagnetic resonance (EPR) spectroscopic characterization of mono-, bi-, and trimolecular RNA structures shows that Çm will be applicable for advanced EPR studies to elucidate structural and dynamic aspects of folded RNA.

A triple spin-labeling strategy coupled with DEER analysis to detect DNA modifications and enzymatic repair.

In a spin: Spin-labeled oligonucleotides produced by click chemistry can be studied by EPR, by using a DEER sequence. This was used to test a complex triple-labeling strategy with damaged DNA. Extensive and accurate analysis of DNA structure and enzymatic repair processes were performed after digestion by EndoIV. Modified DNA structures and DNA-protein interactions can now be readily studied.