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Many viruses contain surface spikes or protrusions that are essential for virus entry. These surface structures can thereby be targeted by antiviral drugs to treat viral infections. Nervous necrosis virus (NNV), a simple nonenveloped virus in the genus of betanodavirus, infects fish and damages aquaculture worldwide. NNV has 60 conspicuous surface protrusions, each comprising three protrusion domains (P-domain) of its capsid protein. NNV uses protrusions to bind to common receptors of sialic acids on the host cell surface to initiate its entry via the endocytic pathway. However, structural alterations of NNV in response to acidic conditions encountered during this pathway remain unknown, while detailed interactions of protrusions with receptors are unclear. Here, we used cryo-EM to discover that Grouper NNV protrusions undergo low-pH-induced compaction and resting. NMR and molecular dynamics (MD) simulations were employed to probe the atomic details. A solution structure of the P-domain at pH 7.0 revealed a long flexible loop (amino acids 311-330) and a pocket outlined by this loop. Molecular docking analysis showed that the N-terminal moiety of sialic acid inserted into this pocket to interact with conserved residues inside. MD simulations demonstrated that part of this loop converted to a β-strand under acidic conditions, allowing for P-domain trimerization and compaction. Additionally, a low-pH-favored conformation is attained for the linker connecting the P-domain to the NNV shell, conferring resting protrusions. Our findings uncover novel pH-dependent conformational switching mechanisms underlying NNV protrusion dynamics potentially utilized for facilitating NNV entry, providing new structural insights into complex NNV-host interactions with the identification of putative druggable hotspots on the protrusion.

The study of cellular machineries responsible for the iron-sulfur (Fe-S) cluster biogenesis has led to the identification of a large number of proteins, whose importance for life is documented by an increasing number of diseases linked to them. The labile nature of Fe-S clusters and the transient protein-protein interactions, occurring during the various steps of the maturation process, make their structural characterization in solution particularly difficult. Paramagnetic nuclear magnetic resonance (NMR) has been used for decades to characterize chemical composition, magnetic coupling, and the electronic structure of Fe-S clusters in proteins; it represents, therefore, a powerful tool to study the protein-protein interaction networks of proteins involving into iron-sulfur cluster biogenesis. The optimization of the various NMR experiments with respect to the hyperfine interaction will be summarized here in the form of a protocol; recently developed experiments for measuring longitudinal and transverse nuclear relaxation rates in highly paramagnetic systems will be also reviewed. Finally, we will address the use of extrinsic paramagnetic centers covalently bound to diamagnetic proteins, which contributed over the last twenty years to promote the applications of paramagnetic NMR well beyond the structural biology of metalloproteins. [ABSTRACT FROM AUTHOR]

The synthesis of tetramethoxyresorcinarene podands bearing p‐toluene arms connected by ‐SO3‐ (1) and ‐CH2O‐ (2) linkers is presented herein. In the solid state, the resorcinarene podand 1 forms an intramolecular self‐inclusion complex with the pendant p‐toluene group of a podand arm, whereas the resorcinarene podand 2 does not show self‐inclusion. The conformations of the flexible resorcinarene podands in solution were investigated by variable‐temperature experiments using 1D and 2D NMR spectroscopic techniques as well as by computational methods, including a conformational search and subsequent DFT optimisation of representative structures. The 1H NMR spectra of 1 and 2 at room temperature show a single set of proton signals that are in agreement with C4v symmetry. At low temperatures, the molecules exist as a mixture of boat conformations featuring slow exchange on the NMR timescale. Energy barriers (ΔG≠298) of 55.5 and 52.0 kJ mol−1 were calculated for the boat‐to‐boat exchange of 1 and 2, respectively. The results of the ROESY experiments performed at 193 K and computational modelling suggest that in solution the resorcinarene podand 1 adopts a similar conformation to that present in its crystal structure, whereas podand 2 populates a more versatile range of conformations in solution. [ABSTRACT FROM AUTHOR]

The study of cellular machineries responsible for the iron–sulfur (Fe–S) cluster biogenesis has led to the identification of a large number of proteins, whose importance for life is documented by an increasing number of diseases linked to them. The labile nature of Fe–S clusters and the transient protein–protein interactions, occurring during the various steps of the maturation process, make their structural characterization in solution particularly difficult. Paramagnetic nuclear magnetic resonance (NMR) has been used for decades to characterize chemical composition, magnetic coupling, and the electronic structure of Fe–S clusters in proteins; it represents, therefore, a powerful tool to study the protein–protein interaction networks of proteins involving into iron–sulfur cluster biogenesis. The optimization of the various NMR experiments with respect to the hyperfine interaction will be summarized here in the form of a protocol; recently developed experiments for measuring longitudinal and transverse nuclear relaxation rates in highly paramagnetic systems will be also reviewed. Finally, we will address the use of extrinsic paramagnetic centers covalently bound to diamagnetic proteins, which contributed over the last twenty years to promote the applications of paramagnetic NMR well beyond the structural biology of metalloproteins.

The crystal structures of the 4-halogenated (hal: F, Cl, Br)-4-butyl-1-phenyl-1,3-pyrolidine-dione (mofebutazone) are determined, and compared with their solution structures. The racemic 4-halogenated mofebutazone approximants crystallize in a monoclinic space group with four molecules in the unit cell. The 4-hal-mofebutazone molecules reveal strong hydrogen bonding between the hydrogen atom located at the N-2 nitrogen atom and a carbonyl oxygen atom of an adjacent 4-hal-mofebutazone molecule. The hydrogen bond angle for 4-Br-mifebutazone N (2) H (1)⋯O (1) is 173(3) °, so that the hydrogen bond is essentially linear indicating an infinite chain hydrogen bond network. The 3d and 2d structures are stabilized by π-π and σ-π interactions, short intermolecular distances, and apolar forces between adjacently stacked phenyl rings. Small-angle-X-ray scattering (SAXS) experiments and osmometric measurements reveal the presence of dimers for the 4-hal-mofebutazone molecules. Molecular simulations indicate similar solution structure factors for the 4-hal-mofebutazones solutions, S(Q), and in the solid state. There is a strong indication that the [1,1,0], [1,0,0], and [1,0,0] periodicities of the 4-Br , 4-Cl and 4-F-mofebutazone in the crystalline solid state were also present in the solution phase. The biochemical and cellular activities of the different 4-hal-mofebutazones were monitored by the magnitude of their inhibition of the PGE 2 biosynthesis through the cyclo-oxygenase (COX-1) in macrophages, and on the inhibition of LTD 4 (5-lipoxygenase) in polymorphonuclear leukocytes. [ABSTRACT FROM AUTHOR]

The high-resolution NMR structure of the first heterocyclic, non-amide, organic cation that strongly and selectively recognizes mixed AT/GC bp (bp=base pair) sequences of DNA in a 1:1 complex is described. Compound designs of this type provide essential methods for control of functional, non-genomic DNA sequences and have broad cell uptake capability, based on studies from animals to humans. The high-resolution structural studies described in this report are essential for understanding the molecular basis for the sequence-specific binding as well as for new ideas for additional compound designs for sequence-specific recognition. The molecular features, in this report, explain the mechanism of recognition of both A⋅T and G⋅C bps and are an interesting molecular recognition story. Examination of the experimental structure and the NMR restrained molecular dynamics model suggests that recognition of the G⋅C base pair involves two specific H-bonds. The structure illustrates a wealth of information on different DNA interactions and illustrates an interfacial water molecule that is a key component of the complex. [ABSTRACT FROM AUTHOR]

Three molecular structures, each containing three different lanthanide(III) centres, have been prepared by coupling three kinetically inert lanthanide(III) complexes in an Ugi reaction. These 2 kDa molecules were purified by dialysis and characterised by NMR and luminescence techniques. The photophysical properties of these heterotrimetallic complexes were investigated and are discussed by comparison with simpler, but related, heterodimetallic compounds. It was found that an aminonaphthalene unit inhibits the sensitisation of terbium, and that the spatial arrangement of the chromophores and lanthanide(III) centres in these molecules inhibits the efficient sensitisation of europium. We conclude that the intramolecular collisions required for efficient Dexter energy transfer from the sensitiser to the lanthanide(III) centre can be prevented by steric congestion. [ABSTRACT FROM AUTHOR]

Bleomycins are a family of glycopeptide antibiotics that have the ability to bind and degrade DNA when bound to key metal ions, which is believed to be responsible for their antitumor activity. Knowledge of the structures of metallo-bleomycins is vital to further characterize their mechanism of action. To this end, numerous structural studies on metallo-bleomycins have been conducted. NMR spectroscopy has had a key role in most of these studies, and has led to very important findings involving the coordination chemistry of metallo-bleomycins, and the details of many metallo-bleomycin-DNA spatial correlations for this important drug. This paper reviews the most important contributions of NMR to the bleomycin field.

Bleomycins are a family of glycopeptide antibiotics that have the ability to bind and degrade DNA when bound to key metal ions, which is believed to be responsible for their antitumor activity. Knowledge of the structures of metallo-bleomycins is vital to further characterize their mechanism of action. To this end, numerous structural studies on metallo-bleomycins have been conducted. NMR spectroscopy has had a key role in most of these studies, and has led to very important findings involving the coordination chemistry of metallo-bleomycins, and the details of many metallo-bleomycin-DNA spatial correlations for this important drug. This paper reviews the most important contributions of NMR to the bleomycin field. [ABSTRACT FROM AUTHOR]

Two sets of terpolymers, polymer A and polymer B consisting of almost same level of 2‐acrylamido‐2‐methyl‐1‐propane sulfonic acid (AMPS) component at 0.635 ± 0.005 m and varying dodecyl methacrylate (DoDMAc) and monomethoxy poly(ethylene glycol) acrylate (PEGAc) components have been designed. Polymer A, consisting of less C12 component, has been shown to promote intermolecular aggregated structures wherein C12 domains exhibit compact packing characteristics. It is demonstrated that in polymer B, AMPS segments are predominantly present as ionic clusters contributing to a high pKa at about 9.50 for a low α of 0.20. From the results of interfacial adsorption estimations at air/solution and water/hexane interface, it is shown that open coil structures provided under high pH (>9.0) conditions promote efficiency of adsorption. This is shown from higher surface excess concentration (Γ) and lower interfacial area (a) estimated using Gibbs adsorption isotherm equation. For example, at water/hexane interface, polymer A exhibits Γ of 1.20 × 10−3 moles/1000 m2 at pH 3.2 and 1.97 × 10−3 moles/1000 m2 at pH 10.0. Significantly, in case of polymer B consisting of ionic clusters of AMPS, adsorption at the liquid/liquid interface is more efficient in comparison to polymer A at all pH. © 2008 Wiley Periodicals, Inc. JPolym Sci Part A: Polym Chem 46: 3257–3271, 2008 [ABSTRACT FROM AUTHOR]

Abstract: Nudix hydrolases catalyze the hydrolysis of nucleoside diphosphates linked to other moieties, X, and contain the sequence motif or Nudix box, GX5EX7REUXEEXGU. The mechanisms of Nudix hydrolases are highly diverse in the position on the substrate at which nucleophilic substitution occurs, and in the number of required divalent cations. While most proceed by associative nucleophilic substitutions by water at specific internal phosphorus atoms of a diphosphate or polyphosphate chain, members of the GDP-mannose hydrolase sub-family catalyze dissociative nucleophilic substitutions, by water, at carbon. The site of substitution is likely determined by the positions of the general base and the entering water. The rate accelerations or catalytic powers of Nudix hydrolases range from 109- to 1012-fold. The reactions are accelerated 103–105-fold by general base catalysis by a glutamate residue within, or beyond the Nudix box, or by a histidine beyond the Nudix box. Lewis acid catalysis, which contributes ⩾103–105-fold to the rate acceleration, is provided by one, two, or three divalent cations. One divalent cation is coordinated by two or three conserved residues of the Nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand from beyond the Nudix box. Some Nudix enzymes require one (MutT) or two additional divalent cations (Ap4AP), to neutralize the charge of the polyphosphate chain, to help orient the attacking hydroxide or oxide nucleophile, and/or to facilitate the departure of the anionic leaving group. Additional catalysis (10–103-fold) is provided by the cationic side chains of lysine and arginine residues and by H-bond donation by tyrosine residues, to orient the general base, or to promote the departure of the leaving group. The overall rate accelerations can be explained by both independent and cooperative effects of these catalytic components. [Copyright &y& Elsevier]

Abstract: Water soluble surface active comb like terpolymers consisting of 0.66:0.22:0.12m (A) and 0.37:0.52:0.11m (B) of 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS), dodecylmethacrylate (DoDMAc) and poly(ethylene glycol) monomethacrylate (PEGAc) macromonomer have been designed. The molecular weights of the polymers A and B are estimated to be 9.64×104 and 9.52×104. It is demonstrated that polymer A promotes intermolecular aggregated structures in contrast to polymer B favoring isolated micellar assemblies. The compact coil structures from polymer B effect considerable reduction in interfacial tension of octadecane/water interface to 14.6mNm-1. [Copyright &y& Elsevier]

Proteins and nucleic acids, alone and in complex are among the essential building blocks of living organisms. Obtaining a molecular level understanding of their structures, and the changes that occur as they interact, is critical for expanding our knowledge of life processes or disease progression. Here, we motivate and describe an application of solution small angle X-ray scattering (SAXS) which provides valuable information about the structures, ensembles, compositions and dynamics of protein-nucleic acid complexes in solution, in equilibrium and time-resolved studies. Contrast variation (CV-) SAXS permits the visualization of the distinct molecular constituents (protein and/or nucleic acid) within a complex. CV-SAXS can be implemented in two modes. In the simplest, the protein within the complex is effectively rendered invisible by the addition of an inert contrast agent at an appropriate concentration. Under these conditions, the structure, or structural changes of only the nucleic acid component of the complex can be studied in detail. The second mode permits observation of both components of the complex: the protein and the nucleic acid. This approach requires the acquisition of SAXS profiles on the complex at different concentrations of a contrast agent. Here, we review CV-SAXS as applied to protein-nucleic acid complexes in both modes. We provide some theoretical framework for CV-SAXS but focus primarily on providing the necessary information required to implement a successful experiment including experimental design, sample quality assessment, and data analysis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The program DYANA, for calculation of solution structures of biomolecules with an algorithm based on simulated annealing by torsion angle dynamics, has been supplemented with a new routine, PSEUDYANA, that enables efficient use of pseudocontact shifts as additional constraints in structure calculations of paramagnetic metalloproteins. PSEUDYANA can determine the location of the metal ion inside the protein frame and allows to define a single tensor of magnetic susceptibility from a family of conformers. As an illustration, a PSEUDYANA structure calculation is provided for a metal-undecapeptide complex, where simulated pseudocontact shifts but no NOE restraints are used as conformational constraints. [ABSTRACT FROM AUTHOR]

Keywords: inclusion compounds; resorcinarenes; solution structures; supramolecular chemistry; X-ray diffraction EN inclusion compounds resorcinarenes solution structures supramolecular chemistry X-ray diffraction 1 1 1 06/12/20 20200610 NES 200610 B Does my crystal structure match with the solution conformation? b A conformationally flexible resorcinarene macrocycle forms a self-inclusion complex in the solid state and adopts a similar folded conformation also in solution. Inclusion compounds, resorcinarenes, solution structures, supramolecular chemistry, X-ray diffraction. [Extracted from the article]