Unconventional surface plasmon resonance signals reveal quantitative inhibition of transcriptional repressor EthR by synthetic ligands

EthR is a mycobacterial repressor that limits the bioactivation of ethionamide, a commonly used anti-tuberculosis second-line drug. Several efforts have been deployed to identify EthR inhibitors abolishing the DNA-binding activity of the repressor. This led to the demonstration that stimulating the bioactivation of Eth through EthR inhibition could be an alternative way to fight Mycobacterium tuberculosis. We propose a new surface plasmon resonance (SPR) methodology to study the affinity between inhibitors and EthR. Interestingly, the binding between inhibitors and immobilized EthR produced a dose-dependent negative SPR signal. We demonstrate that this signal reveals the affinity of small molecules for the repressor. The affinity constants (K_D) correlate with their capacity to inhibit the binding of EthR to DNA. We hypothesize that conformational changes in EthR during ligand interaction could be responsible for this SPR signal. Practically, this unconventional result opens perspectives onto the development of an SPR assay that would at the same time reveal structural changes in the target upon binding with an inhibitor and the binding constant of this interaction.     

Structure of EthR in a ligand bound conformation reveals therapeutic perspectives against tuberculosis

Mycobacterium tuberculosis EthR is a repressor of ethA, a gene encoding a mono-oxygenase required for the activation of the prodrug ethionamide. Here we describe the X-ray crystal structure of EthR, a homodimer with an entirely helical structure showing similarities to TetR family members. Each monomer contained a fortuitous ligand identified as hexadecyl octanoate. The crystal structure of EthR purified in M. smegmatis revealed the presence of a comparable ligand. The binding of hexadecyl octanoate to EthR induces a conformational state incompatible with repressor function, which should lead to ethA derepression and consequently to an increased sensitivity to ethionamide and other thioamides. A related, more hydrophilic ketone was found to exhibit synergistic antimycobacterial effects when tested together with ethionamide, indicating that this strategy may help reduce the dosage of potent antibacterial compounds that otherwise are too toxic to be used as first-line drugs.     

Fast methionine-based solution structure determination of calcium-calmodulin complexes

Here we present a novel NMR method for the structure determination of calcium-calmodulin (Ca²⁺-CaM)-peptide complexes from a limited set of experimental restraints. A comparison of solved CaM-peptide structures reveals invariability in CaM’s backbone conformation and a structural plasticity in CaM’s domain orientation enabled by a flexible linker. Knowing this, the collection and analysis of an extensive set of NOESY spectra is redundant. Although RDCs can define CaM domain orientation in the complex, they lack the translational information required to position the domains on the bound peptide and highlight the necessity of intermolecular NOEs. Here we employ a specific isotope labeling strategy in which the role of methionine in CaM-peptide interactions is exploited to collect these critical NOEs. By ¹H, ¹³C-labeling the methyl groups of deuterated methionine against a ²H, ¹²C background, we can acquire a ¹³C-edited NOESY characterized by simplified, easily analyzable spectra. Together with measured CaM backbone H_N-N RDCs and intrapeptide NOE-based distances, these intermolecular NOEs provide restraints for a low temperature torsion-angle dynamics and simulated annealing protocol used to calculate the complex structure. We have applied our method to a CaM complex previously solved through X-ray crystallography: Ca²⁺-CaM bound to the CaM kinase I peptide (PDB code: 1MXE). The resulting structure has a backbone RMSD of 1.6 Å to that previously published. We have also used this test complex to investigate the importance of homologous model selection on the calculated outcome. In addition to having application for fast complex structure determination, this method can be used to determine the structures of difficult complexes characterized by chemical shift overlap and broad signals for which the traditional method based on the use of fully ¹³C, ¹⁵N-labeled CaM fails.     

Heteroassociation of antibiotic norfloxacin with aromatic vitamins in aqueous solution

Heteroassociation of antibacterial antibiotic norfloxacin with aromatic vitamins nicotinamide and flavin mononucleotide in aqueous solution was studied by ¹H NMR spectroscopy (500 MHz). Equilibrium constants, induced proton chemical shifts, and thermodynamic parameters (ΔH, ΔS) for the reactions of heteroassociation of the molecules were determined on the basis of the concentration and temperature dependences of proton chemical shifts for interacting aromatic molecules. The analysis of the results obtained indicates the formation of heterocomplexes between vitamin molecules and norfloxacin owing to stacking interactions between aromatic chromophores and additional intermolecular hydrogen bonding in norfloxacin-nicotinamide. The most probable spatial structures of 1:1 norfloxacin-flavin mononucleotide and norfloxacin-nicotinamide heterocomplexes were determined by molecular modeling methods using X-PLOR software on the basis of analysis of induced proton chemical shifts.     

Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis

Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral. We report herein that high-throughput chemical experiments can isolate an RNA’s multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M²) and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states’ structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M²-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10%) and conservative error estimates. In experimental benchmarks, M²-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M²-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.     

Superposition of two tRNA acceptor stem crystal structures: Comparison of structure, ligands and hydration

We solved the X-ray structures of two Escherichia coli tRNA^Ser acceptor stem microhelices. As both tRNAs are aminoacylated by the same seryl-tRNA-synthetase, we performed a comparative structure analysis of both duplexes to investigate the helical conformation, the hydration patterns and magnesium binding sites. It is well accepted, that the hydration of RNA plays an important role in RNA-protein interactions and that the extensive solvent content of the minor groove has a special function in RNA. The detailed comparison of both tRNA^Ser microhelices provides insights into the structural arrangement of the isoacceptor tRNA aminoacyl stems with respect to the surrounding water molecules and may eventually help us to understand their biological function at atomic resolution.     

NMR resonance assignments for the class II GTP binding RNA aptamer in complex with GTP

The structures of RNA-aptamer-ligand complexes solved in the last two decades were instrumental in realizing the amazing potential of RNA for forming complex tertiary structures and for molecular recognition of small molecules. For GTP as ligand the sequences and secondary structures for multiple families of aptamers were reported which differ widely in their structural complexity, ligand affinity and ligand functional groups involved in RNA-binding. However, for only one of these families the structure of the GTP-RNA complex was solved. In order to gain further insights into the variability of ligand recognition modes we are currently determining the structure of another GTP-aptamer—the so-called class II aptamer—bound to GTP using NMR-spectroscopy in solution. As a prerequisite for a full structure determination, we report here ¹H, ¹³C, ¹⁵N and partial ³¹P-NMR resonance assignments for the class II GTP-aptamer bound to GTP.     

Structural determination of complex natural products by quantum mechanical calculations of <Superscript>13</Superscript>C NMR chemical shifts: development of a parameterized protocol for terpenes

Nuclear magnetic resonance (NMR) spectroscopy is one of the most important tools for determining the structures of organic molecules. Despite the advances made in this technique, revisions of erroneously established structures for natural products are still commonly published in the literature. In this context, the prediction of chemical shifts through ab initio and density functional theory (DFT) calculations has become a very powerful tool for assisting with the structural determination of complex organic molecules. In this work, we present the development of a protocol for ¹³C chemical shift calculations of terpenes, a class of natural products that are widely distributed among plant species and are very important due to their biological and pharmacological activities. This protocol consists of GIAO-DFT calculations of chemical shifts and the application of a parameterized scaling factor in order to ensure accurate structural determination of this class of natural products. The application of this protocol to a set of five terpenes yielded accurate calculated chemical shifts, showing that this is a very attractive tool for the calculation of complex organic structures such as terpenes.     

Mg2+ Impacts the Twister Ribozyme through Push-Pull Stabilization of Nonsequential Phosphate Pairs

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg²⁺, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg²⁺ in folded conformations of RNA have been studied extensively; however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate, and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/molecular dynamics simulations are used to capture the energetics and atomic-level details of Mg²⁺-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg²⁺, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg²⁺ with two or more nonsequential phosphate groups. Notably, these results indicate a push-pull mechanism in which the Mg²⁺-RNA interactions actually lead to destabilization of specific nonsequential phosphate-phosphate interactions (i.e., pushed apart), whereas other interactions are stabilized (i.e., pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister, including the formation of hydrogen bonds associated with the tertiary structure. This study establishes a better understanding of how Mg²⁺-ion interactions contribute to RNA structural properties and stability.     

Structure-based investigations of the NAD+-II riboswitch

Riboswitches are conserved non-coding domains in bacterial mRNA with gene regulation function that are essential for maintaining enzyme co-factor metabolism. Recently, the pnuC RNA motif was reported to selectively bind nicotinamide adenine dinucleotide (NAD⁺), defining a novel class of NAD⁺ riboswitches (NAD⁺-II) according to phylogenetic analysis. To reveal the three-dimensional architecture and the ligand-binding mode of this riboswitch, we solved the crystal structure of NAD⁺-II riboswitch in complex with NAD⁺. Strikingly and in contrast to class-I riboswitches that form a tight recognition pocket for the adenosine diphosphate (ADP) moiety of NAD⁺, the class-II riboswitches form a binding pocket for the nicotinamide mononucleotide (NMN) portion of NAD⁺ and display only unspecific interactions with the adenosine. We support this finding by an additional structure of the class-II RNA in complex with NMN alone. The structures define a novel RNA tertiary fold that was further confirmed by mutational analysis in combination with isothermal titration calorimetry (ITC), and 2-aminopurine-based fluorescence spectroscopic folding studies. Furthermore, we truncated the pnuC RNA motif to a short RNA helical scaffold with binding affinity comparable to the wild-type motif to allude to the potential of engineering the NAD⁺-II motif for biotechnological applications.     

Unique 31P Spectral Response to the Formation of a Specific Restriction Enzyme–DNA Complex

Protein-induced distortion is a dramatic but not universally observed feature of sequence-specific DNA interactions. This is illustrated by the crystal structures of restriction enzyme–DNA complexes: While some of these structures exhibit DNA distortion, others do not. Among the latter is PvuII endonuclease, a small enzyme that is also amenable to NMR spectroscopic studies. Here ³¹P NMR spectroscopy is applied to demonstrate the unique spectral response of DNA to sequence-specific protein interactions. The ³¹P NMR spectrum of a noncognate DNA exhibits only spectral broadening upon the addition of enzyme. However, when enzyme is added to target DNA, a number of ³¹P resonances shift dramatically. The magnitudes of the chemical shifts (2–3 ppm) are among the largest observed. Site-specific substitution with phosphoramidates and phosphorothioates are used analyze these effects. While such spectral features have been interpreted as indicative of DNA backbone distortions, FRET analysis indicates that this does not occur in PvuII-cognate DNA complexes in solution. The distinct ³¹P spectral signature observed for cognate DNA mirrors that observed for the enzyme, underscoring the unique features of cognate complex formation.     

Interaction between aromatic antibiotics and vitamins: 1H NMR study of heteroassociation of nicotinamide and anthracycline antitumor antibiotics

The interaction of the anthracycline antitumor antibotics daunomycin and novatrone with the vitamin nicotinamide has been studied by one-and two-dimensional ¹H NMR (500 MHz). Due to significant differences between the structures of the chromophores of interacting molecules, a two-site heteroassociation model has been developed, which implies the binding of one or several nicotinamide molecules to the chromophore of the antibiotic. The structural and thermodynamic parameters of the heteroassociation of nicotinamide with daunomycin and novatrone have been determined from the experimental concentration and temperature dependences of the ¹H NMR chemical shifts of the interacting molecules. The most favorable structures of the 1:1 nicotinamide-daunomycin and nicotinamide-novatrone heterocomplexes have been found using the molecular mechanics method (X-PLOR software) and analysis of induced proton chemical shifts. The results demonstrate that two nicotinamide molecules cannot simultaneously bind on one side of the chromophore of the daunomycin or novatrone. The 1:1 heterocomplexes of the vitamin with the antibiotics are mainly stabilized by the stacking of aromatic chromophores.     

A Structural and Thermodynamic Analysis of Novatrone and Flavin Mononucleotide Heteroassociation in Aqueous Solution by <Superscript>1</Superscript>H NMR Spectroscopy

A heteroassociation of the antitumor antibiotic novatrone (NOV) and flavin mononucleotide (FMN) in aqueous solution was studied by one- and two-dimentional ¹H NMR spectroscopy (500 MHz) to elucidate the molecular mechanism of the possible combined action of the antibiotic and the vitamin. The equilibrium reaction constants, the induced proton chemical shifts, and the thermodynamic parameters (ΔH and Δ_S) of the NOV and FMN heteroassociation were determined from the concentration and temperature dependences of proton chemical shifts of the aromatic molecules. The most favorable structure of the 1 : 1 NOV-FMN complex was determined by both the method of molecular mechanics (X-PLOR software) and the induced proton chemical shifts of the molecules. An analysis of the results suggests that the NOV-FMN intermolecular complexes are mainly stabilized by stacking interactions of their aromatic chromophores. An additional stabilization is possible due to intermolecular hydrogen bonds. It was concluded that the aromatic molecules of vitamins, in particular, FMN, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous solutions, which could result in a modulation of their medical and biological action.     

A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide complex of d(CGTACG)(2)

The structures of the complexes formed between 9-amino-[N-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG^5BrUACG)₂ and d(CGTACG)₂ have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 Å. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native).     

Thioamide substitution to probe the hydroxyproline recognition of VHL ligands

Thioamide substitution influences hydrogen bond and n → π¹ interactions involved in the conformational stability of protein secondary structures and oligopeptides. Hydroxyproline is the key recognition element of small molecules targeting the von Hippel-Lindau (VHL) E3 ligase, which are of interest as probes of hypoxia signaling and ligands for PROTAC conjugation. We hypothesized that VHL ligands could be a privileged model system to evaluate the contribution of these interactions to protein:ligand complex formation. Herein we report the synthesis of VHL ligands bearing thioamide substitutions at the central hydroxyproline moiety, and characterize their binding by fluorescence polarization, isothermal titration calorimetry, X-ray crystallography and molecular modeling. In spite of a conserved binding mode, the substitution pattern had a pronounced impact on the ligand affinities. Together the results underscore the role of hydrogen bond and n → π¹ interactions in fine tuning hydroxyproline recognition by VHL.     

MHC Class I/peptide interactions: Binding specificity and kinetics

Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class l molecules as well as in the applications of real time biosensor technology have permitted the direct analysis of the binding of MHC class l molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran-modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as ‘pepsicles’, as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class l peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three-dimensional molecular models of MHC/peptide complexes. Details analysis of the kinetic dissociation rates (k_d) of the MHC molecules from the specifically coupled solid phase peptides revels that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured k_d values for antigenic peptide/class I interactions at 25°C are in the range of ca 10^?4–10^?6/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid-phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.