Solution structure of a cyanovirin-N:Man alpha 1-2Man alpha complex: structural basis for high-affinity carbohydrate-mediated binding to gp120.

BACKGROUND: Cyanovirin-N (CVN) is a novel, 11 kDa cyanobacterial protein that potently inhibits viral entry by diverse strains of HIV through high-affinity carbohydrate-mediated interactions with the viral envelope glycoprotein gp120. CVN contains two symmetry-related carbohydrate binding sites of differing affinities that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinities, the carbohydrates that also mediate CVN:gp120 binding. High-resolution structural studies of CVN in complex with a representative oligosaccharide are desirable for understanding the structural basis for this unprecedented specificity. RESULTS: We have determined by multidimensional heteronuclear NMR spectroscopy the three-dimensional solution structure of CVN in complex with two equivalents of the disaccharide Manalpha1-2Manalpha, a high-affinity ligand which represents the terminal-accessible disaccharide present in Man(8) D1D3 and Man(9). The structure reveals that the bound disaccharide adopts the stacked conformation, thereby explaining the selectivity for Man(8) D1D3 and Man(9) over other oligomannose structures, and presents two novel carbohydrate binding sites that account for the differing affinities of the two sites. The high-affinity site comprises a deep pocket that nearly envelops the disaccharide, while the lower-affinity site comprises a semicircular cleft that partially surrounds the disaccharide. The approximately 40 A spacing of the two binding sites provides a simple model for CVN:gp120 binding. CONCLUSIONS: The CVN:Manalpha1-2Manalpha complex provides the first high-resolution structure of a mannose-specific protein-carbohydrate complex with nanomolar affinity and presents a new carbohydrate binding motif, as well as a new class of carbohydrate binding protein, that facilitates divalent binding via a monomeric protein.

The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinity: implications for binding to the HIV envelope protein gp120.

Cyanovirin-N (CVN) is a monomeric 11 kDa cyanobacterial protein that potently inactivates diverse strains of human immunodeficiency virus (HIV) at the level of cell fusion by virtue of high affinity interactions with the surface envelope glycoprotein gp120. Several lines of evidence have suggested that CVN-gp120 interactions are in part mediated by N-linked complex carbohydrates present on gp120, but experimental evidence has been lacking. To this end we screened a comprehensive panel of carbohydrates which represent structurally the N-linked carbohydrates found on gp120 for their ability to inhibit the fusion-blocking activity of CVN in a quantitative HIV-1 envelope-mediated cell fusion assay. Our results show that CVN specifically recognizes with nanomolar affinity Man(9)GlcNAc(2) and the D1D3 isomer of Man(8)GlcNAc(2). Nonlinear least squares best fitting of titration data generated using the cell fusion assay show that CVN binds to gp120 with an equilibrium association constant (K(a)) of 2.4 (+/- 0.1) x 10(7) M(-1) and an apparent stoichiometry of 2 equiv of CVN per gp120, Man(8)GlcNAc(2) D1D3 acts as a divalent ligand (2 CVN:1 Man(8)) with a K(a) of 5.4 (+/- 0.5) x 10(7) M(-1), and Man(9)GlcNAc(2) functions as a trivalent ligand (3 CVN:1 Man(9)) with a K(a) of 1.3 (+/- 0.3) x 10(8) M(-1). Isothermal titration calorimetry experiments of CVN binding to Man(9)GlcNAc(2) at micromolar concentrations confirmed the nanomolar affinity (K(a) = 1.5 (+/- 0.9) x 10(8) M(-1)), and the fitted data indicated a stoichiometry equal to approximately one (1 Man(9):1 CVN). The 1:1 stoichiometry at micromolar concentrations suggested that CVN has not only a high affinity binding site-relevant to the studies at nM concentrations-but a lower affinity site as well that facilitates cross-linking of CVN-oligomannose at micromolar concentrations or higher. The specificity of CVN for Man(8) D1D3 and Man(9) over the D1D2 isomer of Man(8) indicated that the minimum structure required for high affinity binding comprises Manalpha1 --> 2Manalpha. By following the (1)H-(15)N correlation spectrum of (15)N-labeled CVN upon titration with this disaccharide, we unambiguously demonstrate that CVN recognizes and binds to the disaccharide Manalpha1 --> 2Manalpha via two distinct binding sites of differing affinities located on opposite ends of the protein. The high affinity site has a K(a) of 7.2 (+/- 4) x 10(6) M(-1) and the low affinity site a K(a) of 6.8 (+/- 4) x 10(5) M(-1) as determined by isothermal titration calorimetry. Mapped surfaces of the carbohydrate binding sites are presented, and implications for binding to gp120 are discussed.

Novel bromotyrosine alkaloids: inhibitors of mycothiol S-conjugate amidase.

[structure: see text] The novel alkaloids 1 and 4 were isolated from an Australian non-verongid sponge, Oceanapia sp. Compound 1 contains an unprecedented imidazolyl-quinolinone substructure attached to a bromotyrosine-derived spiro-isoxazoline. Three other known alkaloids were isolated in addition to 1 and 4 and together represent the first examples of inhibitors of a new mycobacterial enzyme mycothiol S-conjugate amidase (MCA).

Design and properties of N(CCG)-gp41, a chimeric gp41 molecule with nanomolar HIV fusion inhibitory activity.

The design and characterization of a chimeric protein, termed N(CCG)-gp41, derived from the ectodomain of human immunodeficiency virus (HIV), type I gp41 is described. N(CCG)-gp41 features an exposed trimeric coiled-coil comprising the N-terminal helices of the gp41 ectodomain. The trimeric coiled-coil is stabilized both by fusion to a minimal thermostable ectodomain of gp41 and by engineered intersubunit disulfide bonds. N(CCG)-gp41 is shown to inhibit HIV envelope-mediated cell fusion at nanomolar concentrations with an IC(50) of 16.1 +/- 2.8 nm. It is proposed that N(CCG)-gp41 targets the exposed C-terminal region of the gp41 ectodomain in its pre-hairpin intermediate state, thereby preventing the formation of the fusogenic form of the gp41 ectodomain, which comprises a highly stable trimer of hairpins arranged in a six-helix bundle. N(CCG)-gp41 has potential as a therapeutic agent for the direct inhibition of HIV cell entry, as an anti-HIV vaccine, and as a component of a rapid throughput assay for screening for small molecule inhibitors of HIV envelope-mediated cell fusion. It is anticipated that antibodies raised against N(CCG)-gp41 may target the trimeric coiled-coil of N-terminal helices of the gp41 ectodomain that is exposed in the pre-hairpin intermediate state in a manner analogous to peptides derived from the C-terminal helix of gp41 that are currently in clinical trials.

Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping.

The crystal structure of cyanovirin-N (CV-N), a protein with potent antiviral activity, was solved at 1.5 A resolution by molecular replacement using as the search model the solution structure previously determined by NMR. The crystals belong to the space group P3221 with one monomer of CV-N in each asymmetric unit. The primary structure of CV-N contains 101 residues organized in two domains, A (residues 1 to 50) and B (residues 51 to 101), with a high degree of internal sequence and structural similarity. We found that under the conditions of the crystallographic experiments (low pH and 26 % isopropanol), two symmetrically related monomers form a dimer by domain swapping, such that domain A of one monomer interacts with domain B' of its crystallographic symmetry mate and vice versa. Because the two swapped domains are distant from each other, domain swapping does not result in additional intramolecular interactions. Even though one of the protein sample solutions that was used for crystallization clearly contained 100 % monomeric CV-N molecules, as judged by various methods, we were only able to obtain crystals containing domain-swapped dimers. With the exception of the unexpected phenomenon of domain swapping, the crystal structure of CV-N is very similar to the NMR structure, with a root-mean-square deviation of 0.55 A for the main-chain atoms, the best agreement reported to date for structures solved using both techniques.

Minor groove-binding architectural proteins: structure, function, and DNA recognition.

To date, high-resolution structures have been solved for five different architectural proteins complexed to their DNA target sites. These include TATA-box-binding protein, integration host factor (IHF), high mobility group I(Y)[HMG I(Y)], and the HMG-box-containing proteins SRY and LEF-1. Each of these proteins interacts with DNA exclusively through minor groove contacts and alters DNA conformation. This paper reviews the structural features of these complexes and the roles they play in facilitating assembly of higher-order protein-DNA complexes and discusses elements that contribute to sequence-specific recognition and conformational changes.

Solution structure of cyanovirin-N, a potent HIV-inactivating protein.

The solution structure of cyanovirin-N, a potent 11,000 Mr HIV-inactivating protein that binds with high affinity and specificity to the HIV surface envelope protein gp120, has been solved by nuclear magnetic resonance spectroscopy, including extensive use of dipolar couplings which provide a priori long range structural information. Cyanovirin-N is an elongated, largely beta-sheet protein that displays internal two-fold pseudosymmetry. The two sequence repeats (residues 1-50 and 51-101) share 32% sequence identity and superimpose with a backbone atomic root-mean-square difference of 1.3 A. The two repeats, however, do not form separate domains since the overall fold is dependent on numerous contacts between them. Rather, two symmetrically related domains are formed by strand exchange between the two repeats. Analysis of surface hydrophobic clusters suggests the location of potential binding sites for protein-protein interactions.

The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif.

The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51-90), and a DNA dodecamer containing the PRDII site of the interferon-beta promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contracts. The absence of one of these modules in the third DNA binding domain accounts for its-100 fold reduced affinity relative to the second one.

Design of an expression system for detecting folded protein domains and mapping macromolecular interactions by NMR.

Two protein expression vectors have been designed for the preparation of NMR samples. The vectors encode the immunoglobulin-binding domain of streptococcal protein G (GB1 domain) linked to the N-terminus of the desired proteins. This fusion strategy takes advantage of the small size, stable fold, and high bacterial expression capability of the GB1 domain to allow direct NMR spectroscopic analysis of the fusion protein by 1H-15N correlation spectroscopy. Using this system accelerates the initial assessment of protein NMR projects such that, in a matter of days, the solubility and stability of a protein can be determined. In addition, 15N-labeling of peptides and their testing for DNA binding are facilitated. Several examples are presented that demonstrate the usefulness of this technique for screening protein/DNA complexes, as well as for probing ligand-receptor interactions, using 15N-labeled GB1-peptide fusions and unlabeled target.

Two classes of metabolites from Theonella swinhoei are localized in distinct populations of bacterial symbionts.

The marine sponge Theonella swinhoei (lithistid Family Theonellidae, Order Astrophorida) has yielded many important, bioactive natural products, most of which share structural features with bacterial natural products. The presence of microbial symbionts in T. swinhoei has been reported, and it was originally suggested that the cytotoxic macrolide swinholide A and many of the bioactive cyclic peptides from T. swinhoei were all produced by simbiotic cyanobacteria. By transmission electron microscopy, we found four distinct cell populations to be consistently present in T. swinhoei: eukaryotic sponge cells, unicellular heterotrophic bacteria, unicellular cyanobacteria and filamentous heterotrophic bacteria. Purification and chemical analyses of each cell type showed the macrolide swinholide A to be limited to the mixed population of unicellular heterotrophic bacteria, and an anti-fungal cyclic peptide occurred only in the filamentous heterotrophic bacteria. Contrary to prior speculation, no major metabolites were located in the cyanobacteria or sponge cells.

The structure of U17 isolated from Streptomyces clavuligerus and its properties as an antioxidant thiol.

The predominant low-molecular-mass thiol produced by streptomycetes is a cysteine derivative previously designated as U17 [Newton, G. L., Fahey, R. C., Cohen, G. & Aharonowitz, Y. (1993) J. Bacteriol. 175, 2734-2742]. In this study we report the elucidation of the structure of the monobromobimane derivative of U17, which establishes the structure of U17 as 2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranosyl-myo-inositol. The presence of the N-acetylcysteine moiety was indicated by formation of N-acetylcysteine-monobromobimane during acid hydrolysis of the monobromobimane derivative of U17. Complete hydrolysis released 1 mol glucosamine/mol cysteine as determined by carbohydrate and amino acid analysis. High-resolution mass spectral analysis gave a precise mass consistent with the molecular formula C27H40N4O14S. Analysis of 13C-NMR, one-dimensional 1H-NMR and two-dimensional NMR experiments identified the remaining C6H12O6 moiety as myo-inositol, confirmed the presence of N-acetylcysteine and glucosamine, and established the connectivity of the components. Two chemical properties of this novel thiol make it suitable as an intracellular storage form of cysteine and as an antioxidant thiol. First, it undergoes heavy-metal-ion catalyzed autoxidation at a rate dramatically lower than that for cysteine and markedly lower than that for glutathione or N-acetylcysteine. Secondly, the alpha-(1-->1) glycosidic link between glucosamine and myo-inositol is resistant to acid hydrolysis, hydrolysing at a rate comparable to that of the two amide bonds in the molecule.

Violence in pregnancy.

Statistics available in the UK and in North America indicate that violence towards women by their partner is widespread but under-reported. Research shows that violence is likely to begin or escalate during pregnancy. It is suggested that violence to pregnant women may be more common than gestational diabetes or placenta praevia, and that it may be responsible for miscarriage, preterm labour and fetal injuries. This paper examines attitudes prevalent in society towards abused women, considers the psychological consequences of their abuse and the inherent difficulties women face when they try to leave a relationship with a violent partner. It also describes how midwives may recognise women who have been abused by their partner and suggests the development of strategies to help such women.