Contryphan-Vn: a modulator of Ca2+-dependent K+ channels

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.     

Surface changes and role of buried water molecules during the sulfane sulfur transfer in rhodanese from Azotobacter vinelandii: a fluorescence quenching and nuclear magnetic relaxation dispersion spectroscopic study

The Azotobacter vinelandii rhodanese is a sulfurtransferase enzyme that catalyzes the transfer of the outer sulfur atom from thiosulfate to cyanide. Recently, investigations by NMR relaxation on the (15)N-enriched protein reported that interdomain contacts are rigidly maintained upon the sulfane sulfur transfer from the enzyme to the substrate. The modality of the enzymatic mechanism is then confined to a surface interaction, including dynamics of water molecules buried in the tertiary structure. Thus, investigations have been carried out by fluorescence, circular dichroism, and nuclear magnetic relaxation dispersion measurements. The comparison of circular dichroism spectra of the persulfurated enzyme and the sulfur-free form indicated that small changes occur. Fluorescence quenching studies have been performed to evaluate the conformational changes during catalysis using the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid, and acrylamide, iodide, and cesium ions as quenchers. Changes in exchange dynamics of water molecules buried in the structure with bulk water, observed by nuclear magnetic relaxation dispersion, are due to local conformational transitions, likely involving residues around the active site, and are consistent with the global correlation time found by (15)N relaxation. These results, taken together, provide important information for elucidating the conformational features of the mechanism of action of the enzyme either in the role of a selective donor of a sulfur atom to small-sized substrates (i.e., to cyanide, transforming it into thiocyanate) or in the role of sulfur insertase for the formation of the Fe(2)S(2) iron-sulfur cluster in sulfur-deprived ferredoxins.     

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.     

PIKfyve controls fluid phase endocytosis but not recycling/degradation of endocytosed receptors or sorting of procathepsin D by regulating multivesicular body morphogenesis

The mammalian phosphatidylinositol (PtdIns) 5-P/PtdIns 3,5-P2-producing kinase PIKfyve has been implicated in maintaining endomembrane homeostasis in mammalian cells. To address the role of PIKfyve in trafficking processes, we examined the functioning of the biosynthetic, endocytic, and recycling pathways in stable human embryonic kidney 293 cell lines inducibly expressing the wild-type or kinase-defective dominant-negative form. PIKfyveWT or PIKfyveK1831E expression did not affect the processing and lysosomal targeting of newly synthesized procathepsin D. Likewise the rates of transferrin uptake/recycling or epidermal growth factor receptor degradation were not altered upon expression of either protein. In contrast, PIKfyveK1831E but not PIKfyveWT expression markedly impaired the late uptake of fluid phase marker horseradish peroxidase. Inspection of the organelle morphology by confocal microscopy with specific markers in COS cells transiently expressing PIKfyveK1831E showed the Golgi apparatus, end lysosomes, and the recycling compartment indistinguishable from nontransfected cells, despite the dramatic PIKfyveK1831E-induced endomembrane vacuolation. In contrast, we observed a striking effect on the late endocytic compartment, marked by disruption of the dextran-labeled perinuclear endosomal compartment and formation of dispersed enlarged vesicles. Electron microscopy identified the cytoplasmic vacuoles in the PIKfyveK1831E-expressing human embryonic kidney 293 cells as enlarged multivesicular body-like structures with substantially lower number of internal vesicles and membrane whorls. Together, these data indicate that PIKfyve selectively regulates the sorting and traffic of peripheral endosomes containing lysosomaly directed fluid phase cargo through controlling the morphogenesis and function of multivesicular bodies.     

Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of Lipid A fatty acid distribution

Lipid A is the lipophilic moiety of lipopolysaccharides (LPSs), the major components of the external membrane of almost all gram-negative bacteria. It is responsible for the toxicity of LPS and has a heterogeneous structure composed of a bis-phosphorylated glucosamine disaccharide backbone that is acylated at the positions 2, 3 of the GlcN I (proximal) and GlcN II (distal) residue with O- and N-linked 3-hydroxy fatty acids (primary substitution). These fatty acids are further acylated by means of their 3-hydroxy groups (secondary substitution). The toxicity of Lipid A is dependent on its primary structure; the number, the length, and the distribution of the fatty acids on the disaccharide backbone strongly influence the endotoxic activity. In this paper a general and easy methodology to obtain secondary fatty acid distribution, which is one of the most difficult issues in the structural determination of Lipid A, is proposed. The method combines ammonium hydroxide hydrolysis and matrix assisted laser desorption ionization (MALDI)-mass spectrometry analysis and has been successfully proven with five different Lipid A species. The procedure exploits the lower stability under mild alkaline conditions of acyl and acyloxyacyl esters with respect to that of the acyl and acyloxyacyl amides. The partially degraded Lipid A species obtained are analyzed by MALDI-MS. The generality of this approach was tested on five Lipid As, namely those arising from Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas reactans, and Burkholderia caryophylli.     

Mechanical unfolding of a titin Ig domain: structure of unfolding intermediate revealed by combining AFM,molecular dynamics simulations,NMR and protein engineering

The mechanical unfolding of an immunoglobulin domain from the human muscle protein titin (TI I27) has been shown to proceed via a metastable intermediate in which the A-strand is detached. The structure and properties of this intermediate are characterised in this study. A conservative destabilising mutation in the A-strand has no effect on the unfolding force, nor the dependence of the unfolding force on the pulling speed, indicating that the unfolding forces measured in an AFM experiment are those required for the unfolding of the intermediate and not the native state. A mutant of TI I27 with the A-strand deleted (TI I27-A) is studied by NMR and standard biophysical techniques, combined with protein engineering. Molecular dynamics simulations show TI I27-A to be a good model for the intermediate. It has a structure very similar to the native state, and is surprisingly stable. Comparison with a Phi-value analysis of the unfolding pathway clearly shows that the protein unfolds by a different pathway under an applied force than on addition of denaturant.     

Structural determination of the complex exopolysaccharide from the virulent strain of Cryphonectria parasitica

The structure of a new exopolysaccharide from the virulent strain of Cryphonectria parasitica was elucidated by means of 2D NMR spectroscopy and selective degradations (mild hydrolysis and acetolysis). The polysaccharide is built up of mannose, galactose and rhamnose and has a rather complex non-repetitive structure that can be idealised as follows:     

Solution structure of DAPI selectively bound in the minor groove of a DNA T.T mismatch-containing site: NMR and molecular dynamics studies.

The solution structure of the complex between 4', 6-diamidino-2-phenylindole (DAPI) and DNA oligomer [d(GCGATTCGC)]2, containing a central T.T mismatch, has been characterized by combined use of proton one- and two-dimensional NMR spectroscopy, molecular mechanics and molecular dynamics computations including relaxation matrix refinement. The results show that the DAPI molecule binds in the minor groove of the central region 5'-ATT-3' of the DNA oligomer, which predominantly adopts a duplex structure with a global right-handed B-like conformation. In the final models of the complex, the DAPI molecule is located nearly isohelical with its NH indole proton oriented towards the DNA helix axis and forming a bifurcated hydrogen bond with the carbonyl O2 groups of a mismatched T5 and the T6 residue of the opposite strand. Mismatched thymines adopt a wobble base pair conformation and are found stacked between the flanking base pairs, inducing only minor local conformational changes in global duplex structure. In addition, no other binding mechanisms were observed, showing that minor groove binding of DAPI to the mismatch-containing site is favoured in comparison with any other previously reported interaction with G.C sequences.