Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR.

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Nav1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Nav1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Nav1.4. Here, we determine the NMR structure of a selectively 13C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By 13C NMR, we obtain NMR structural constraints as 13C chemical shifts and the 1H-2H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the 1H-2H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel NavRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Nav1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.

Simple and accurate single base resolution analysis of 5-hydroxymethylcytosine by catalytic oxidative bisulfite sequencing using micelle incarcerated oxidants.

Oxidation of 5-methylcytosine (5mC) is catalyzed by ten-eleven translocation (TET) enzymes to produce 5-hydroxymethylcytosine (5hmC) and following oxidative products. The oxidized nucleotides were shown to be the intermediates for DNA demethylation, as the nucleotides are removed by base excision repair system initiated by thymine DNA glycosylase. A simple and accurate method to determine initial oxidation product 5hmC at single base resolution in genomic DNA is necessary to understand demethylation mechanism. Recently, we have developed a new catalytic oxidation reaction using micelle-incarcerated oxidants to oxidize 5hmC to form 5-formylcytosine (5fC), and subsequent bisulfite sequencing can determine the positions of 5hmC in DNA. In the present study, we described the optimization of the catalytic oxidative bisulfite sequencing (coBS-seq), and its application to the analysis of 5hmC in genomic DNA at single base resolution in a quantitative manner. As the oxidation step showed quite low damage on genomic DNA, the method allows us to down scale the sample to be analyzed.

Crystal Structure of Okadaic Acid Binding Protein 2.1: A Sponge Protein Implicated in Cytotoxin Accumulation.

Okadaic acid (OA) is a marine polyether cytotoxin that was first isolated from the marine sponge Halichondria okadai. OA is a potent inhibitor of protein serine/threonine phosphatases (PP) 1 and 2A, and the structural basis of phosphatase inhibition has been well investigated. However, the role and mechanism of OA retention in the marine sponge have remained elusive. We have solved the crystal structure of okadaic acid binding protein 2.1 (OABP2.1) isolated from H. okadai; it has strong affinity for OA and limited sequence homology to other proteins. The structure revealed that OABP2.1 consists of two α-helical domains, with the OA molecule deeply buried inside the protein. In addition, the global fold of OABP2.1 was unexpectedly similar to that of aequorin, a jellyfish photoprotein. The presence of structural homologues suggested that, by using similar protein scaffolds, marine invertebrates have developed diverse survival systems adapted to their living environments.

Selective oxidation of 5-hydroxymethylcytosine with micelle incarcerated oxidants to determine it at single base resolution.

5-Methylcytosine (5mC) is oxidized by ten-eleven translocation (TET) enzymes. This process followed by thymine DNA glycosylase is proposed to be the mechanism for methylcytosine demethylation. 5-Hydroxymethylcytosine (5hmC) is one of the most important key oxidative metabolites in the demethylation process, and therefore, simple and accurate method to determine 5hmC at single base resolution is desired. In the present study, we developed a mild catalytic oxidation of 5-hmC using micelle incarcerated oxidants that enables to determine the position of 5hmC at single base resolution.

Truncated norzoanthamine exhibiting similar collagen protection activity, toward a promising anti-osteoporotic drug.

The marine alkaloid, norzoanthamine, is considered to be a promising drug for osteoporosis treatment. Due to its rarity and complicated structure, a practical supply method must be developed. Here, we designed a truncated norzoanthamine, which has two-thirds of the original structure, and found that it exhibited similar collagen protection activity.

Structure-activity relationships of the truncated norzoanthamines exhibiting collagen protection toward anti-osteoporotic activity.

The marine alkaloid norzoanthamine is a candidate drug for osteoporosis treatment. Due to its structural complexity, simplified analogues possessing similar biological activities are needed for further research. Recently, we found that the bisaminal unit, representing two-thirds of the original structure, is a bioactive equivalent. We synthesized three kinds of further truncated norzoanthamines and evaluated their collagen protection activities. No analog with collagen protection activity comparable to that of the bisaminal unit was found. Thus, we confirmed the importance of the bisaminal unit for the collagen protection activity. Furthermore, we found that the recognition tolerance of the substrate collagen is relatively large by comparing both enantiomers.

Interaction analysis of a ladder-shaped polycyclic ether and model transmembrane peptides in lipid bilayers by using Förster resonance energy transfer and polarized attenuated total reflection infrared spectroscopy.

Ladder-shaped polycyclic ethers (LSPs) are predicted to interact with membrane proteins; however, the underlying mechanism has not been satisfactorily elucidated. It has been hypothesized that LSPs possess non-specific affinity to α-helical segments of transmembrane proteins. To verify this hypothesis, we constructed a model LSP interaction system in a lipid bilayer. We prepared 5 types of α-helical peptides and reconstituted them in liposomes. The reconstitution and orientation of these peptides in the liposomes were examined using polarized attenuated total reflection infrared (ATR-IR) spectroscopy and gel filtration. The results revealed that 4 peptides were retained in liposomes, and 3 of them formed stable transmembrane structures. The interaction between the LSP and the peptides was investigated using Förster resonance energy transfer (FRET). In the lipid bilayer, the LSP strongly recognized the peptides that possessed aligned hydrogen donating groups with leucine caps. We propose that this leucine-capped 16-amino acid sequence is a potential LPS binding motif.

Brevisulcenal-F: a polycyclic ether toxin associated with massive fish-kills in New Zealand.

A novel marine toxin, brevisulcenal-F (KBT-F, from karenia brevisulcata toxin) was isolated from the dinoflagellate Karenia brevisulcata. A red tide of K. brevisulcata in Wellington Harbour, New Zealand, in 1998 was extremely toxic to fish and marine invertebrates and also caused respiratory distress in harbor bystanders. An extract of K. brevisulcata showed potent mouse lethality and cytotoxicity, and laboratory cultures of K. brevisulcata produced a range of novel lipid-soluble toxins. A lipid soluble toxin, KBT-F, was isolated from bulk cultures by using various column chromatographies. Chemical investigations showed that KBT-F has the molecular formula C(107)H(160)O(38) and a complex polycyclic ether nature. NMR and MS/MS analyses revealed the complete structure for KBT-F, which is characterized by a ladder-frame polyether scaffold, a 2-methylbut-2-enal terminus, and an unusual substituted dihydrofuran at the other terminus. The main section of the molecule has 17 contiguous 6- and 7-membered ether rings. The LD(50) (mouse i.p.) for KBT-F was 0.032 mg/kg.

Origins of oxygen atoms in a marine ladder-frame polyether: evidence of monooxygenation by 18O-labeling and using tandem mass spectrometry.

Yessotoxin is a ladder-frame polyether produced by the dinoflagellate Protoceratium reticulatum. Previous labeling experiments using (13)C-acetate established the unique assembly of the carbon chain from intact and cleaved acetate units. The origins of ether and hydroxy oxygens in the molecule, which would yield further information regarding the assembly of the ladder-frame structure, have yet to be established. In this study, we describe the incorporation of (18)O in one experiment where the dinoflagellate was cultured under (18)O(2) atmosphere and in a second where the culture media was supplemented with [(18)O(2)]acetate. Labeled yessotoxin obtained from these experiments was subjected to collision-induced dissociation tandem mass spectrometry to determine the positions of (18)O-incorporation pattern in the molecule. Detailed analyses of product ions from the fragmentation processes led to the identification of (18)O-labeled positions and the incorporation ratios. The data revealed that the ether oxygens were labeled from (18)O(2) and the hydroxy oxygen on C32 was derived from [(18)O(2)]acetate. These results support a proposed biosynthetic mechanism of marine ladder-frame polyethers that a polyene precursor was oxidized by a monooxygenase after acetate condensation.

Solution NMR analysis of the binding mechanism of DIVS6 model peptides of voltage-gated sodium channels and the lipid soluble alkaloid veratridine.

Voltage-gated sodium channels (VGSCs) are responsible for generating action potentials in nervous systems. Veratridine (VTD), a lipid soluble alkaloid isolated from sabadilla lily seed, is believed to bind to segment 6 of VGSCs and act as a partial agonist. However, high resolution structural interaction mechanism between VGSCs and VTD is difficult to elucidate because of the large size and membrane localization of VGSCs. Here, the authors designed model peptides corresponding to domain IV segment 6 (DIVS6) of rat skeletal muscle Na(v)1.4 and analyzed the complex of the model peptides and VTD by solution NMR analysis to obtain structural information of the interaction. The model peptides successfully formed an α-helices, which is the suspected native conformation of DIVS6, in aqueous 2,2,2-trifluoroethanol, a membrane-mimicking solvent. The VTD binding residues of the model peptide were identified using the NMR titration experiments with VTD, including a newly discovered VTD binding residue Leu14 (µ1-L1580 in Na(v)1.4), which has not been reported by point mutation studies. Mapping of VTD binding residues on the model peptide revealed the hydrophobic interaction surface. NMR titration experiments with a non-toxic analog of VTD, veracevine, also indicated that the steroidal backbone of VTD interacts with the hydrophobic interaction surface of DIVS6 and that the 3-acyl group of VTD possibly causes neurotoxicity by interacting with domain I segment 6 and/or domain IV segment 4.

Prorocentrol, a polyoxy linear carbon chain compound isolated from the toxic dinoflagellate Prorocentrum hoffmannianum.

A polyoxy linear carbon chain compound, prorocentrol (1), was isolated from cultured cells of the dinoflagellate Prorocentrum hoffmannianum, which produces a polyether carboxylic acid, okadaic acid. The structure of 1 was elucidated by detailed analyses of 2D NMR spectra. Compound 1 possesses 30 hydroxy groups, 1 ketone, and 8 double bonds on the C65-linear carbon chain. Its partial relative configuration was deduced by the proton-proton and long-range carbon-proton coupling constants, and compound 1 showed moderate cytotoxicity and antidiatom activity.

Binding of diarrheic shellfish poisoning toxins to okadaic acid binding proteins purified from the sponge Halichondria okadai.

Okadaic acid (OA) and dinophysistoxin-1 (DTX1) cause diarrheic shellfish poisoning. This article examines the biochemical interactions of the two toxins with novel okadaic acid binding proteins (OABPs) 2.1 and 2.3, originally isolated from the marine sponge Halichondria okadai. First, recombinant OABPs 2.1 and 2.3 were expressed in Escherichia coli BL21 (DE3) cells. Binding assays using [24-(3)H]OA and the recombinant OABP 2.1 or 2.3 demonstrated the dissociation constant K(d) of 1.30±0.56 nM and 1.54±0.35 nM, respectively. Binding of [24-(3)H]okadaic acid to recombinant OABP2.1 was almost equally replaced with OA and DTX1. OA-induced cytotoxicity in mouse leukemia P388 cells was inhibited in the presence of the recombinant OABPs 2.1 and 2.3 with an EC(50) of 92±8.4 nM and 87±13 nM, respectively. These results suggest that the blockage of OA-induced cytotoxicity by OABPs 2.1 and 2.3 may be involved in regulating symbiotic relationships present in the sponge H. okadai.

Computationally and experimentally derived general rules for fragmentation of various glycosyl bonds in sodium adduct oligosaccharides.

Mechanisms of fragmentation of glycosyl bond linkages in various saccharides were investigated by using computational calculations to find general rules of fragmentation of sodiated oligosaccharides in mass spectrometry. The calculations revealed that alpha-Glc, alpha-Gal, beta-Man, alpha-Fuc, beta-GlcNAc, and beta-GalNAc linkages were cleaved more easily than beta-Glc, beta-Gal, and alpha-Man linkages because the transition states of the former were stabilized by the anomeric effect. The 1-6 linkage was more stable than the others, since saccharides with flexible 1-6 linkages were more stabilized in energy than the other linkages by the sodium cation. The sialyl linkage was the most labile of all the linkages investigated. Comparison of activation energies and binding affinities to the sodium cation revealed an increase in activation energy in proportion to the increment in binding affinity. The calculated stabilities of glycosyl bonds were: alpha-Man (Manalpha1-3Man, Manalpha1-4Man, Manalpha1-6Man) > beta-Gal (Galbeta1-4Gal) > alpha-GalNAc (GalNAcalpha1-4GalNAc) > beta-Man (Manbeta1-4GlcNAc) > alpha-Gal (Galalpha1-3Gal, Galalpha1-4Gal, Galalpha1-6Gal) > beta-Man (Manbeta1-4Man) > beta-GalNAc (GalNAcbeta1-4GalNAc) > alpha-Fuc (Fucalpha1-6GlcNAc) > alpha-Fuc (Fucalpha1-4GlcNAc) > beta-GlcNAc (GlcNAcbeta1-4GlcNAc) > alpha-Fuc (Fucalpha1-3GlcNAc) > alpha-NeuNAc (NeuNAcalpha2-3Gal, NeuNAcalpha2-6Gal); this result was close to the experimentally deduced trend. These theoretically and experimentally derived general rules for fragmentation should be useful for analyzing the experimentally obtained mass spectra of oligosaccharides.

N-terminal labeling of proteins by the Pictet-Spengler reaction.

The Pictet-Spengler reaction was applied to the N-terminal labeling of horse heart myoglobin. This was performed in the following two steps: (1) conversion of the N-terminal glycine residue to an alpha-keto aldehyde by a transamination reaction and (2) condensation of the resulting activated myoglobin with tryptamine analogues by the Pictet-Spengler reaction. Ultraviolet (UV)/visible (vis) absorption and circular dichroism (CD) spectral data revealed that the tertiary structure of myoglobin was not altered by the Pictet-Spengler reaction.

Conformations of 3-carboxylic esters essential for neurotoxicity in veratrum alkaloids are loosely restricted and fluctuate.

The lipid-soluble veratrum alkaloids, veratridine and cevadine, are plant neurotoxins that are agonists of voltage-gated sodium channel. Their conformations in a hydrophobic environment were analyzed by NMR spectroscopy in solution phase chloroform at low temperatures. The conformations around the 3-carboxylic esters which is essential for their neurotoxicity, was completely different from the previously reported X-ray crystallographic structure. The carbonyl oxygen atom (O28) of the carboxylic ester forms a weak intramolecular hydrogen bond with the OH proton at C4 (4-OH) that loosely restricts the conformation of the 3-veratroyl ester in veratridine and the 3-angeloyl ester in cevadine. Methylation at C4 hydroxyl group of veratridine had much reduced its neurotoxic activity relating to voltage-gated sodium channel. The results suggested that the loose conformational restrictions of the carboxylic esters are important for neurotoxicity of the veratrum alkaloids.

Solid-phase fluorescence and ionization efficiency in negative-ion matrix-assisted laser desorption/ionization of neutral oligosaccharides: interaction between beta-carboline matrix and ammonium salt.

Ammonium chloride (NH4Cl) with a beta-carboline harmine (7-methoxy-1-methyl-9H-pyrido[3,4-b]indole) Matrix promotes the generation of chloride-anionized molecules of neutral oligosaccharides in negative-ion ultraviolet matrix-assisted laser/desorption ionization mass spectrometry (MALDI MS). The relative abundances of anionized molecules and matrix ([Analyte + Cl]-/[matrix + Cl]-) correlate with the amount of NH4Cl added, and saturate at a level of NH4Cl approximately four times that of the matrix. Their solid-phase fluorescence spectra indicate that harmine and NH4Cl form a complex of the hydrochloride salt in the mixed crystal. The peak intensity at 448 nm from the harmine hydrochloride in the mixed crystal rises logarithmically with the amount of NH4Cl added, a result that quantitatively correlates with increases in the ion abundance ratios of the chloride-anionized molecules to that of harmine. The solid-phase spectroscopic method is useful for studying changes in the characteristics of the matrices and additives in the mixed crystal. Harmine hydrochloride, rather than harmine, works as an effective matrix. The attachment of the chloride to the matrix is essential for the generation of chlorinated-anionized molecules in MALDI. An N-acetyl glucosamine residue (GlcNAc) in lacto-N-tetraose promotes the generation of their chloride-anionized molecules, however, multi-GlcNAc residues in N-acetylchitooligosaccharides hinder it.

A comparative study of the fragmentation of neutral lactooligosaccharides in negative-ion mode by UV-MALDI-TOF and UV-MALDI ion-trap/TOF mass spectrometry.

Structure analyses of underivatized neutral lacto oligosaccharides are systematically performed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) and UV-MALDI ion-trap time-of-flight mass spectrometry (ion-trap/TOF MS) acquired in negative-ion mode. Interestingly, their fragmentation significantly differ each other. In postsource decay (PSD) in UV-MALDI TOF MS, cross-ring cleavage at the reducing terminal predominates. On the other hand, glycosyl bond cleavage (C-type fragmentation) takes place preferentially in collision induced dissociation (CID) in UV-MALDI ion-trap/TOF MS. The cross-ring cleavage in PSD similar to that in in-source decay occurs via a prompt reaction path characteristic of the UV-MALDI process itself. The product ion spectra of UV-MALDI ion-trap/TOF MS are similar to the electrospray ionization (ESI) ion-trap or quadrupole/TOF CID product ion spectra. During ion-trap/TOF MS experiments, the deprotonated molecular ions survive for several tens of milliseconds after CID event because the high internal energy chlorinated precursor ions are cooled by collisional cooling in the ion trap. The results obtained suggest that the PSD from the chlorinated precursor ion in UV-MALDI TOF MS might proceed as a two-step reaction; in the first, a high internal energy deprotonated molecular ion is generated as a reaction intermediate during the flight in the drift tube, and in the second, the rapid decomposition from the deprotonated molecular ion takes place.

Semiquantitative analysis of isomeric oligosaccharides by negative-ion mode UV-MALDI TOF postsource decay mass spectrometry and their fragmentation mechanism study at N-acetyl hexosamine moiety.

Postsource decay (PSD) spectra of isomeric neutral lactooligosaccharide mixtures were measured from the chlorinated molecules [M + Cl]- by negative-ion mode ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) to estimate quantitatively the mixing ratios in their mixtures. The PSD ions specific to each isomeric structure were used to distinguish the linkage and branching isomers, and the molar ratios of the isomers were estimated from their ion abundances. The relative ion abundances changed linearly in the PSD spectra of the mixtures of the isomers as their molar ratio was varied in the analyte solutions. Therefore, the molar ratios of the isomers in the analyte mixtures could be estimated semiquantitatively. In addition, we studied their fragmentation mechanisms in N-acetyl hexosamines such as GlcNAc, which enabled us to quantitatively analyze the structures of the isomers of lactooligosaccharides. The conjugated systems elongate in the chemical species of the Z-type fragmentation on the 3-linked GlcNAc owing to the acetoamido groups at the C-2 positions, which made the chemical species of the Z-type ions stable. The glycosyl bonds of the front of GlcNAc cleaved easily as a C-type fragmentation because the negative charge at the anomeric position could be delocalized to the carbonyl oxygen atom at the acetoamido group of GlcNAc. These factors caused the stabilization of the chemical species of the C/Z fragment ions produced by the double cleavage around GlcNAc.

Identification of okadaic acid binding protein 2 in reconstituted sponge cell clusters from Halichondria okadai and its contribution to the detoxification of okadaic acid.

Okadaic acid (OA) and OA binding protein 2 (OABP2) were previously isolated from the marine sponge Halichondria okadai. Because the amino acid sequence of OABP2 is completely different from that of protein phosphatase 2A, a well-known target of OA, we have been investigating the production and function of OABP2. In the present study, we hypothesized that OABP2 plays a role in the detoxification of OA in H. okadai and that the OA concentrations are in proportional to the OABP2 concentrations in the sponge specimens. Based on the OA concentrations and the OABP2 concentrations in the sponge specimens collected in various places and in different seasons, however, we could not determine a positive correlation between OA and OABP2. We then attempted to determine distribution of OA and OABP2 in the sponge specimen. When the mixture of dissociated sponge cells and symbiotic species were separated with various pore-sized nylon meshes, most of the OA and OABP2 was detected from the same 0-10 µm fraction. Next, when sponge cell clusters were prepared from a mixture of dissociated sponge cells and symbiotic species in the presence of penicillin and streptomycin, we identified the 18S rDNA of H. okadai and the gene of OABP2 in the analysis of genomic DNA but could not detect OA by LC-MS/MS. We thus concluded that the sponge cells express OABP2, and that OA was not apparently present in the sponge cells but could be colocalized with OABP2 in the sponge cells at a concentration less than the limit of detection.

Studies toward the total synthesis of gymnocin A, a cytotoxic polyether: a highly convergent entry to the F-N ring fragment.

[structure: see text] An efficient and highly convergent synthesis of the FGHIJKLMN ring fragment of gymnocin A, a cyctotoxic polycyclic ether isolated from the notorious red-tide forming dinoflagellate Gymnodinium mikimotoi, has been achieved. The present synthesis relied on extensive use of the B-alkyl Suzuki-Miyaura coupling reaction.