Cyclopropane-Based Peptidomimetics Mimicking Wide-Ranging Secondary Structures of Peptides: Conformational Analysis and Their Use in Rational Ligand Optimization.

Detailed conformational analyses of our previously reported cyclopropane-based peptidomimetics and conformational analysis-driven ligand optimization are described. Computational calculations and X-ray crystallography showed that the characteristic features of cyclopropane function effectively to constrain the molecular conformation in a three-dimensionally diverse manner. Subsequent principal component analysis revealed that the diversity covers the broad chemical space filled by peptide secondary structures in terms of both main-chain and side-chain conformations. Based on these analyses, a lead stereoisomer targeting melanocortin receptors was identified, and its potency and subtype selectivity were improved by further derivatization. The presented strategy is effective not only for designing non-peptidic ligands from a peptide ligand but also for the rational optimization of these ligands based on the plausible target-binding conformation without requiring the three- dimensional structural information of the target and its peptide ligands.

Conformational divergence in the HA-33/HA-17 trimer of serotype C and D botulinum toxin complex.

Clostridium botulinum produces a large toxin complex (L-TC) comprising botulinum neurotoxin associated with auxiliary nontoxic proteins. A complex of 33- and 17-kDa hemagglutinins (an HA-33/HA-17 trimer) enhances L-TC transport across the intestinal epithelial cell layer via binding HA-33 to a sugar on the cell surface. At least two subtypes of serotype C/D HA-33 exhibit differing preferences for the sugars sialic acid and galactose. Here, we compared the three-dimensional structures of the galactose-binding HA-33 and HA-33/HA-17 trimers produced by the C-Yoichi strain. Comparisons of serotype C/D HA-33 sequences reveal a variable region with relatively low sequence similarity across the C. botulinum strains; the variability of this region may influence the manner of sugar-recognition by HA-33. Crystal structures of sialic acid- and galactose-binding HA-33 are broadly similar in appearance. However, small-angle X-ray scattering revealed distinct solution structures for HA-33/HA-17 trimers. A structural change in the C-terminal variable region of HA-33 might cause a dramatic shift in the conformation and sugar-recognition mode of HA-33/HA-17 trimer.

Sugar-induced conformational change found in the HA-33/HA-17 trimer of the botulinum toxin complex.

Large-sized botulinum toxin complex (L-TC) is formed by conjugation of neurotoxin, nontoxic nonhemagglutinin and hemagglutinin (HA) complex. The HA complex is formed by association of three HA-70 molecules and three HA-33/HA-17 trimers, comprised of a single HA-17 and two HA-33 proteins. The HA-33/HA-17 trimer isolated from serotype D L-TC has the ability to bind to and penetrate through the intestinal epithelial cell monolayer in a sialic acid-dependent manner, and thus it plays an important role in toxin delivery through the intestinal cell wall. In this study, we determined the solution structure of the HA-33/HA-17 trimer by using small-angle X-ray scattering (SAXS). The SAXS image of HA-33/HA-17 exhibited broadly similar appearance to the crystal image of the complex. On the other hand, in the presence of N-acetylneuraminic acid, glucose and galactose, the solution structure of the HA-33/HA-17 trimer was drastically altered compared to the structure in the absence of the sugars. Sugar-induced structural change of the HA-33/HA-17 trimer may contribute to cell binding and subsequent transport across the intestinal cell layer.

Small-angle X-ray scattering reveals structural dynamics of the botulinum neurotoxin associating protein, nontoxic nonhemagglutinin.

In cell culture supernatants, the botulinum neurotoxin (BoNT) exists as part of a toxin complex (TC) in which nontoxic nonhemagglutinin (NTNHA) and/or hemagglutinins (HAs) are assembled onto the BoNT. A series of investigations indicated that formation of the TC is vital for delivery of the toxin to nerve cells through the digestive tract. In the assembly process, BoNT binds to NTNHA yielding M-TC, and it then matures into L-TC by further association with the HAs via NTNHA in the M-TC. Here, we report a crystal structure of the NTNHA from Clostridium botulinum serotype D strain 4947. Additionally, we performed small-angle X-ray scattering (SAXS) analysis of the NTNHA and the M-TC to elucidate the solution structure. The crystal structure of D-4947 NTNHA revealed that BoNT and NTNHA share a closely related structure consisting of three domains. The SAXS image indicated that, even though the N-terminal two-thirds of the NTNHA molecule had an apparently similar conformation in both the crystal and solution structures, the C-terminal third of the molecule showed a more extended structure in the SAXS image than that seen in the crystallographic image. The discrepancy between the crystal and solution structures implies a high flexibility of the C-terminal third domain of NTNHA, which is involved in binding to BoNT. Structural dynamics of the NTNHA molecule revealed by SAXS may explain its binding to BoNT to form the BoNT/NTNHA complex.

Crystallization and preliminary X-ray analysis of the Clostridium botulinum type D nontoxic nonhaemagglutinin.

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex assembled with nontoxic nonhaemagglutinin (NTNHA) and/or haemagglutinin components. Complex formation with NTNHA is considered to be critical in eliciting food poisoning because the complex shields the BoNT from the harsh conditions in the digestive tract. In the present study, NTNHA was expressed in Escherichia coli and crystallized. Diffraction data were collected to 3.9 Å resolution. The crystal belonged to the trigonal space group P321 or P3(1)21/P3(2)21, with unit-cell parameters a = b = 147.85, c = 229.74 Å. The structure of NTNHA will provide insight into the assembly mechanism that produces the unique BoNT-NTNHA complex.

Epoxyquinone formation catalyzed by a two-component flavin-dependent monooxygenase involved in biosynthesis of the antibiotic actinorhodin.

The biosynthetic gene cluster of the aromatic polyketide antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2) carries a pair of genes, actVA-ORF5 and actVB, that encode a two-component flavin-dependent monooxygenase (FMO). Our previous studies have demonstrated that the ActVA-ORF5/ActVB system functions as a quinone-forming C-6 oxygenase in ACT biosynthesis. Furthermore, we found that this enzyme system exhibits an additional oxygenation activity with dihydrokalafungin (DHK), a proposed intermediate in the ACT biosynthetic pathway, and generates two reaction products. These compounds were revealed to be monooxygenated derivatives of kalafungin, which is spontaneously formed through oxidative lactonization of DHK. Their absolute structures were elucidated from their NMR spectroscopic data and by computer modeling and X-ray crystallography as (5S,14R)-epoxykalafungin and (5R,14S)-epoxykalafungin, demonstrating an additional epoxyquinone-forming activity of the ActVA-ORF5/ActVB system in vitro.

Spontaneous mutagenesis associated with nucleotide excision repair in Escherichia coli.

The vast majority of spontaneous mutations occurring in Escherichia coli are thought to be derived from spontaneous DNA lesions, which include oxidative base damage. Systems for removing intrinsic mutagens and repairing DNA lesions contribute to the suppression of spontaneous mutations. Nucleotide excision repair (NER) is a general DNA repair system that eliminates various kinds of lesions from DNA. We therefore predicted that NER might be involved in suppression of spontaneous mutations, and analyzed base substitutions occurring spontaneously within the rpoB gene in NER-proficient (wild-type), -deficient and -overproducing E. coli strains. Surprisingly, the mutation frequency was lower in NER-deficient strains, and higher in NER-overproducing strains, than in the NER-proficient strain. These results suggest, paradoxically, that NER contributes to the generation of spontaneous mutation rather than to its suppression under normal growth conditions, and that transcription-coupled repair also participates in this process. Using E. coli strains that carried an editing exonuclease-deficient polA mutation, we further obtained data suggesting that unnecessary NER might account for these findings, so that errors introduced during repair DNA synthesis by DNA polymerase I would result in unwanted base substitutions. The repair system itself may thus be an important generator of spontaneous mutation.

Clostridium botulinum serotype D neurotoxin and toxin complex bind to bovine aortic endothelial cells via sialic acid.

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.

Role of nontoxic components of serotype D botulinum toxin complex in permeation through a Caco-2 cell monolayer, a model for intestinal epithelium.

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). To assess the role of nontoxic components in the oral intoxication of botulinum TCs, we investigated the permeability of serotype D strain 4947 BoNT and its various TC species through cultured Caco-2 cell monolayers. The L-TC species (complexes composed of BoNT, NTNHA, HA-70, HA-33 and HA-17) showed potent permeability through the cell layer, whereas free BoNT, M-TC (BoNT and NTNHA complexes) and M-TC/HA-70 showed little or no permeability. Cell binding tests demonstrated that HA-33/HA-17 complexes bound to cells, whereas other components did not. These findings suggest that BoNT in the 650-kDa L-TC permeates into the cell mainly in an HA-33/HA-17-mediated manner, although free BoNT can permeate into the cell. As free BoNT and M-TC were susceptible to digestion with gastrointestinal juice, it is likely that L-TC species containing HA-33 caused higher oral toxicity in mice than others. We conclude that the HA-33 subcomponent plays a critical role in the permeation of TCs into intestinal epithelium, and that other HA subcomponents protect BoNT against gastrointestinal digestion.

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli.

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.

Effect of nicking the C-terminal region of the Clostridium botulinum serotype D neurotoxin heavy chain on its toxicity and molecular properties.

A unique strain of Clostridium botulinum serotype D 4947 produces toxin complexes that are composed of un-nicked components, including a neurotoxin (BoNT) and auxiliary proteins. This BoNT showed aberrant elution upon Superdex gel filtration, indicating a much lower molecular weight, due to hydrophobic interaction with the column. Limited trypsin proteolysis of BoNT produces two nicks; first nick yielded a BoNT 50 kDa light chain disulfide linked to a 100 kDa heavy chain (Hc), and a second nick arose in Hc C-terminal 10 kDa. The second nick occurred in the putative binding domain of the BoNT molecule and induced alterations in its secondary structure, leading to a significant reduction of mouse toxicity in comparison with that of the fully-activated singly nicked BoNT. These results help to clarify the role of the C-terminal half of the Hc in the oral toxicity of single-chain and more complex forms of BoNT.

Building-block architecture of botulinum toxin complex: Conformational changes provide insights into the hemagglutination ability of the complex.

Clostridium botulinum produces the botulinum neurotoxin (BoNT). Previously, we provided evidence for the "building-block" model of botulinum toxin complex (TC). In this model, a single BoNT is associated with a single nontoxic nonhemagglutinin (NTNHA), yielding M-TC; three HA-70 molecules are attached and form M-TC/HA-70, and one to three "arms" of the HA-33/HA-17 trimer (two HA-33 and one HA-17) further bind to M-TC/HA-70 via HA-17 and HA-70 binding, yielding one-, two-, and three-arm L-TC. Of all TCs, only the three-arm L-TC caused hemagglutination. In this study, we determined the solution structures for the botulinum TCs using small-angle X-ray scattering (SAXS). The mature three-arm L-TC exhibited the shape of a "bird spreading its wings", in contrast to the model having three "arms", as revealed by transmission electron microscopy. SAXS images indicated that one of the three arms of the HA-33/HA-17 trimer bound to both HA-70 and BoNT. Taken together, these findings regarding the conformational changes in the building-block architecture of TC may explain why only three-arm L-TC exhibited hemagglutination.

Quantitative detection of gene expression and toxin complex produced by Clostridium botulinum serotype D strain 4947.

Botulinum toxin is produced by Clostridium botulinum as a large toxin complex (L-TC) non-covalently assembled with a neurotoxin (NT), a non-toxic non-hemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33, and HA-17). In this study, the gene expressions of five individual L-TC components were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in C. botulinum serotype D strain 4947 (D-4947) during cell growth. Transcripts for the five component genes were successfully detected in the mid-exponential growth phase (6.5 h), reaching a maximum at the early stationary growth phase (12 h). The ratio of the mRNA transcripts of nt and ntnha was approximately 1:1, suggesting that nt and ntnha are bicistronically transcribed. On the other hand, the transcript levels of the ha genes were several-fold higher than those of nt and ntnha, although the mRNA transcript level of ha-33 was less than the other two ha subcomponent genes. The results based on qRT-PCR indicate that a shortage of HA-33 among the proteins associated with botulinum TC could explain the production by D-4947 of other smaller-sized L-TCs (610, 540 and 410 kDa) with fewer HA-33 molecules than the mature 650 kDa L-TC. Western blot analysis demonstrated that TC species in cell lysate were initially observed in the mid-exponential phase, while extracellular TCs were detected subsequently in the early stationary phase.

Characterization of toxin complex produced by a unique strain of Clostridium botulinum serotype D 4947.

A unique strain of Clostridium botulinum, serotype D 4947 (D-4947), produces a considerable amount of a 650 kDa toxin complex (L-TC) and a small amount of a 280 kDa M-TC, a 540 kDa TC, and a 610 kDa TC. The complexes are composed of only un-nicked components, including neurotoxin (NT), nontoxic nonhemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33 and HA-17). Unlike other NTs from all serotype strains, separation of D-4947 NT from L-TC, except for M-TC, during chromatography required highly alkaline conditions around pH 8.8. The separated NT and NTNHA/HAs complex can be reconstituted to L-TC that is indistinguishable from the parent L-TC with respect to toxicity, hemagglutination activity and gel filtration profile. The isoelectric points of NT and NTNHA/HAs were close together depending on the number of HA-33/17 molecules. We have established a new method to separate the unique D-4947 NT from the complex, which will yield valuable information on structure of botulinum toxin.

Organocatalyzed regio- and enantioselective allylic trifluoromethylation of Morita-Baylis-Hillman adducts using Ruppert-Prakash reagent.

The organocatalyzed regioselective allylic trifluoromethylation of Morita-Baylis-Hillman adducts using Ruppert-Prakash reagent was achieved in high to excellent yields via a successive S(N)2'/S(N)2' mode for the first time. The reaction was extended to the asymmetric allylic trifluoromethylation by the use of a bis-cinchona alkaloid catalyst with high enantioselectivities up to 94% ee.

Highly fluorescent M2L4 molecular capsules with anthracene shells.

M(2)L(4) molecular capsules self-assembled from M(II) ions (where M = Zn, Ni, and Pd) and bent bidentate ligands constructed from anthracene fluorophores. The Ni(II) and Zn(II) capsules exhibited weak to strong blue emission unlike traditional Pd(II) cages and capsules.

Biyoulactones A-C, new pentacyclic meroterpenoids from Hypericum chinense.

Three novel pentacyclic meroterpenoids with a unique dilactone structure containing C-C bonded bi- and tricyclic γ-lactone moieties, biyoulactones A-C (1-3), were isolated from the roots of Hypericum chinense, and their structures were elucidated on the basis of spectroscopic data. The relative and absolute stereochemistry of 1 was assigned by a combination of NOESY and a single crystal X-ray diffraction analysis.

Redox-responsive molecular helices with highly condensed π-clouds.

Helices have long attracted the attention of chemists, both for their inherent chiral structure and their potential for applications such as the separation of chiral compounds or the construction of molecular machines. As a result of steric forces, polymeric o-phenylenes adopt a tight helical conformation in which the densely packed phenylene units create a highly condensed π-cloud. Here, we show an oligomeric o-phenylene that undergoes a redox-responsive dynamic motion. In solution, the helices undergo a rapid inversion. During crystallization, however, a chiral symmetry-breaking phenomenon is observed in which each crystal contains only one enantiomeric form. Crystals of both handedness are obtained, but in a non-racemic mixture. Furthermore, in solution, the dynamic motion of the helical oligomer is dramatically suppressed by one-electron oxidation. X-ray crystallography of both the neutral and oxidized forms indicated that a hole, generated upon oxidation, is shared by the repeating o-phenylene units. This enables conformational locking of the helix, and represents a long-lasting chiroptical memory.

HA-33 facilitates transport of the serotype D botulinum toxin across a rat intestinal epithelial cell monolayer.

A large size botulinum toxin complex (L-TC) is composed of a single neurotoxin (BoNT), a single nontoxic nonhaemagglutinin (NTNHA) and a haemagglutinin (HA) complex. The HA complex is comprised of three HA-70 molecules and three arm structures of HA-33/HA-17 that consist of two HA-33 and a single HA-17. In addition to the mature L-TC, smaller TCs are present in cultures: M-TC (BoNT/NTNHA), M-TC/HA-70 and immature L-TCs with fewer HA-33/HA-17 arms than mature L-TC. Because L-TC displays higher oral toxicity than pure BoNT, it was presumed that nontoxic proteins are critical for food poisoning. In this study, the absorption of TCs across intestinal epithelial cells was assessed by examining the cell binding and monolayer transport of serotype D toxins in the rat intestinal epithelial cell line IEC-6. All TCs, including pure BoNT, displayed binding and transport, with mature L-TC showing the greatest potency. Inhibition experiments using antibodies revealed that BoNT, HA-70 and HA-33 could be responsible for the binding and transport. The findings here indicate that all TCs can transport across the cell layer via a sialic acid-dependent process. Nonetheless, binding and transport markedly increased with number of HA-33/HA-17 arms in the TC. We therefore conclude that the HA-33/HA-17 arm is not necessarily required for, but facilitates, transport of botulinum toxin complexes.