Hikaru Hemmi

Solution structure of moricin, an antibacterial peptide, isolated from the silkworm Bombyx mori.

A novel antibacterial peptide, moricin, isolated from the silkworm Bombyx mori, consists of 42 amino acids. It is highly basic and the amino acid sequence has no significant similarity to those of other antibacterial peptides. The 20 structures of moricin in methanol have been determined from two‐dimensional 1H‐nuclear magnetic resonance spectroscopic data. The solution structure reveals an unique structure comprising of a long α‐helix containing eight turns along nearly the full length of the peptide except for four N‐terminal residues and six C‐terminal residues. The electrostatic surface map shows that the N‐terminal segment of the α‐helix, residues 5–22, is an amphipathic α‐helix with a clear separation of hydrophobic and hydrophilic faces, and that the C‐terminal segment of the α‐helix, residues 23–36, is a hydrophobic α‐helix except for the negatively charged surface at the position of Asp30. The results suggest that the amphipathic N‐terminal segment of the α‐helix is mainly responsible for the increase in permeability of the membrane to kill the bacteria.

NMR studies on the interaction of sugars with the C-terminal domain of an R-type lectin from the earthworm Lumbricus terrestris.

The R‐type lectin EW29, isolated from the earthworm Lumbricus terrestris, consists of two homologous domains (14 500 Da) showing 27% identity with each other. The C‐terminal domain (Ch; C‐half) of EW29 (EW29Ch) has two sugar‐binding sites in subdomains α and γ, and the protein uses these sugar‐binding sites for its function as a single‐domain‐type hemagglutinin. In order to determine the sugar‐binding ability and specificity for each of the two sugar‐binding sites in EW29Ch, ligand‐induced chemical‐shift changes in EW29Ch were monitored using 1H–15N HSQC spectra as a function of increasing concentrations of lactose, melibiose, d‐galactose, methyl α‐d‐galactopyranoside and methyl β‐d‐galactopyranoside. Shift perturbation patterns for well‐resolved resonances confirmed that all of these sugars associated independently with the two sugar‐binding sites of EW29Ch. NMR titration experiments showed that the sugar‐binding site in subdomain α had a slow or intermediate exchange regime on the chemical‐shift timescale (Kd = 10−2 to 10−1 mm), whereas that in subdomain γ had a fast exchange regime for these sugars (Kd = 2–6 mm). Thus, our results suggest that the two sugar‐binding sites of EW29Ch in the same molecule retain its hemagglutinating activity, but this activity is 10‐fold lower than that of the whole protein because EW29Ch has two sugar‐binding sites in the same molecule, one of which has a weak binding mode.

Structure determination of a siderophore peucechelin from Streptomyces peucetius

Previously, Park et al. isolated a new siderophore from Streptomyces peucetius ATCC 27952 based on information of the genome sequence and the structure of the siderophore was deduced to be a cyclic peptide based on MS/MS analysis. To clarify the structure of the siderophore, we cultured S. peucetius with iron deficient medium. Through several chromatographic procedures, the siderophore named peucechelin was isolated with the yield enough to perform NMR experiments. The planar structure of peucechelin was elucidated by the combination of ESI-MS experiment and NMR spectroscopic analyses of the gallium (III) complex. Unlike the previously deduced cyclic structure, the structure was determined to be a linear peptide which was similar to a known siderophore foroxymithine. The stereochemistries of amino acids constituting peucechelin were determined by applying modified Marfey method to the hydrolysate. Since the biosynthetic gene of peucechelin was formerly determined by Park et al. the similar genes were searched using genome data of other streptomycetes. As a result, the similar genes were found in the genome data of S. venezuelae and S. purpureus. Isolation and identification of siderophore was performed from the iron deficient culture of S. venezuelae. The siderophore of S. venezuelae was identified to be known compound foroxymithine by analysis ESI-MS and NMR spectra in the similar manner with peucechelin. Production of foroxymithine was also observed in the iron deficient culture of S. purpureus. Based on the genome data, comparison of the biosynthetic genes of structurally related siderophores peucechelin and foroxymithine was accomplished in discussion.

Structure, epitope mapping, and docking simulation of a gibberellin mimic peptide as a peptidyl mimotope for a hydrophobic ligand

Using NMR spectroscopy and simulated annealing calculations, we determined the solution structure of the disulfide‐linked cyclized decapeptide ACLPWSDGPC (SD), which is bound to an anti‐(gibberellin A4) mAb 4‐B8(8)/E9 and was found to be the first peptidyl mimotope for a hydrophobic ligand. The resulting structure of the peptide showed a β‐turn‐like conformation in residues three to seven and the region converges well (average rmsd 0.54 Å). The binding activity and the epitopes of the peptide to the antibody were assessed using saturation transfer difference (STD)‐NMR experiments. We also conducted docking simulations between the peptide and the mAb to determine how the peptide is bound to the mAb. Resonances around the β‐turn‐like conformation of peptide SD (residues 3–5) showed strong STD enhancement, which agreed well with results from docking simulation between peptide SD and the mAb. Together with the commonality of amino acid residues of the mAb involved in interactions with gibberellin A4 (GA4) and peptide SD, we concluded that peptide SD is bound to the antigen‐binding site of mAb 4‐B8(8)/E9 as a GA4 mimic, confirming evidence for the existence of peptide mimics even for hydrophobic ligands.

NMR structure and dynamics of the C-terminal domain of R-type lectin from the earthworm Lumbricus terrestris

The C‐terminal domain (Ch; C‐half) of the R‐type earthworm 29‐kDa lectin (EW29), isolated from the earthworm Lumbricus terrestris, has two sugar‐binding sites, in subdomains α and γ, and the protein uses the two sugar‐binding sites for its function as a single domain‐type haemagglutinin. Our previous NMR titration experiments showed that the α sugar‐binding site is a high‐affinity site and the γ sugar‐binding site is a low‐affinity site. However, it remains unclear why the α sugar‐binding site of EW29Ch binds to lactose much more strongly because the crystal structure of lactose‐bound EW29Ch showed that the interaction between the α sugar‐binding site and lactose was almost same as that between the γ sugar‐binding site and lactose. In the present study, we have determined the NMR structure of EW29Ch in the sugar‐free state and performed 15N relaxation experiments for EW29Ch in both the sugar‐free state and the lactose‐bound states. The conformation of EW29Ch in the sugar‐free state was similar to that of EW29Ch in complex with lactose. Conformational changes upon binding of lactose were observed only for the α sugar‐binding site. By contrast, the 15N relaxation experiments revealed a conformational exchange at the α sugar‐binding site in the sugar‐free state, which was suppressed in the lactose‐bound state. The conformational exchange phenomenon observed for the α sugar‐binding site was not observed for the γ sugar‐binding site. Differences in the conformational change and the backbone dynamics between subdomains α and γ may be associated with the difference of the sugar‐binding modes between the two sugar‐binding sites.

Isolation and structural determination of a new hydrophobic peptide venepeptide from Streptomyces venezuelae

Isolation and structural determination of a new hydrophobic peptide venepeptide from Streptomyces venezuelae

Inversion of the stereochemistry around the sulfur atom of the axial methionine side chain through alteration of amino acid side chain packing in Hydrogenobacter thermophilus cytochrome C552 and its functional consequences.

In cytochrome c, the coordination of the axial Met Sδ atom to the heme Fe atom occurs in one of two distinctly different stereochemical manners, i.e., R and S configurations, depending upon which of the two lone pairs of the Sδ atom is involved in the bond; hence, the Fe-coordinated Sδ atom becomes a chiral center. In this study, we demonstrated that an alteration of amino acid side chain packing induced by the mutation of a single amino acid residue, i.e., the A73V mutation, in Hydrogenobacter thermophilus cytochrome c552 (HT) forces the inversion of the stereochemistry around the Sδ atom from the R configuration [Travaglini-Allocatelli, C., et al. (2005) J. Biol. Chem. 280, 25729-25734] to the S configuration. Functional comparison between the wild-type HT and the A73V mutant possessing the R and S configurations as to the stereochemistry around the Sδ atom, respectively, demonstrated that the redox potential (Em) of the mutant at pH 6.00 and 25 °C exhibited a positive shift of ∼20 mV relative to that of the wild-type HT, i.e., 245 mV, in an entropic manner. Because these two proteins have similar enthalpically stabilizing interactions, the difference in the entropic contribution to the Em value between them is likely to be due to the effect of the conformational alteration of the axial Met side chain associated with the inversion of the stereochemistry around the Sδ atom due to the effect of mutation on the internal mobility of the loop bearing the axial Met. Thus, the present study demonstrated that the internal mobility of the loop bearing the axial Met, relevant to entropic control of the redox function of the protein, is affected quite sensitively by the contextual stereochemical packing of amino acid side chains in the proximity of the axial Met.

Isolation and structure determination of a new cytotoxic peptide, curacozole, from Streptomyces curacoi based on genome mining

Using genome mining, a new cytotoxic peptide named curacozole was isolated from Streptomyces curacoi. Through ESI-MS and NMR analyses, curacozole was determined to be a macrocyclic peptide containing two isoleucine, two thiazole and three oxazole moieties. Curacozole exhibited potent cytotoxic activity against HCT116 and HOS cancer cells. The proposed biosynthetic gene cluster of curacozole was identified and compared with that of the related compound YM-216391.

Streptopeptolin, a Cyanopeptolin-Type Peptide from Streptomyces olivochromogenes

Cyanopeptolin-type peptides are cyclic depsipeptides that commonly have 3-amino-6-hydroxy-2-piperidone (Ahp) unit in the molecules. So far, cyanopeptolin-type peptides have been isolated as protease inhibitors from a wide variety of cyanobacteria. In the course of screening for new peptides, a new peptide streptopeptolin, which had the similar structure to cyanopeptolin, was isolated from the extract of Streptomyces olivochromogenes NBRC 3561. Streptopeptolin is the first cyanopeptolin-type peptide isolated from actinobacteria. The structure of streptopeptolin was determined by the analysis of electrospray ionization mass spectrometry and NMR to be cyclic depsipeptide containing unusual amino acids, Ahp, and N-methyl tyrosine. As a result of protease inhibition test, streptopeptolin showed inhibitory activity against chymotrypsin. The whole genome sequence data of S. olivochromogenes revealed the biosynthetic gene cluster for streptopeptolin, which encoded a nonribosomal peptide synthetase. We proposed a biosynthetic pathway of streptopeptolin based on bioinformatics analysis.

Isolation and structural determination of a new antibacterial compound demethyl-L-681,217 from Streptomyces cattleya

Isolation and structural determination of a new antibacterial compound demethyl-L-681,217 from Streptomyces cattleya