Structural and functional studies of LaIT2, an antimicrobial and insecticidal peptide from Liocheles australasiae.

LaIT2, composed of 59 amino acid residues, is a peptide toxin isolated from the venom of the Yaeyama scorpion, Liocheles australasiae. LaIT2 is toxic to insects but not most mammals. The N- and C-domains of LaIT2 are known to possess antimicrobial and insecticidal activities, respectively. However, the molecular mechanisms are largely unknown because of the lack of a three-dimensional structure of LaIT2. Thus, we elucidated the solution NMR structure of LaIT2. LaIT2 adopts a ß-KTx-like two-domain structure, in which the N- and C-terminal domains form a random coil and an α-ß-ß motif, respectively. Trifluoro ethanol and liposomes titration experiments showed that the unstructured N-domain of LaIT2 has the ability to form an α-helix. The N-terminal helix is amphiphilic, and one side of the helix is positively charged. Measurements of the antimicrobial and insecticidal activities of LaIT2 mutants suggested K15 in the N-domain was found to be responsible for the antimicrobial activities, whereas L53 and L54 in the C-domain were key residues involved in the insecticidal activity. Moreover, K21 in the N-domain is important for both activities. Therefore, two domains are suggested that they work together to show antimicrobial and insecticidal activity.

Overexpression and purification of a toxic peptide LaIT2 from Japanese scorpion, Liocheles australasiae.

In Japan, there are two species of scorpions, Madara scorpion (Isometrus maculatus) and Yaeyama scorpion (Liocheles australasiae), and both of them are living in Yaeyama island. It has been shown that Liocheles australasiae has venom including ß-toxin acting on K+-channels (ß-KTx) (Juichi et al., 2018) [1]. Interestingly, LaIT2, one of the toxins found in the venom of Liocheles australasiae, displays the virulence for insects but almost not for mammals. Until now, molecular mechanism of the functional specificity of LaIT2 is unknown. To clear this issue, we tried to establish the overexpression system of LaIT2 in Rosetta-gami B (DE3) pLysS, which have trxB/gor mutations to induce the disulfide bond formation. In this study, we have succeeded to overexpress the recombinant LaIT2 (rLaIT2) as a thioredoxin (Trx)-tagged protein, and established the purification protocol with Ni2+-NTA column chromatography, enterokinase digestion, and HPLC. We succeeded to obtain approximately 0.5 mg of rLaIT2 from the E. coli cells cultured in 1 L of M9 culture medium. Intramolecular disulfide bonding pattern of rLaIT2 was identified by endopeptidase fragmentation and mass spectrometry. rLaIT2 showed insecticidal activity and antimicrobial activity, and these are almost identical to those of natural LaIT2. 1H-15N HSQC spectrum of 15N-labeled rLaIT2 indicated that the rLaIT2 has a stable conformation.

A novel mechanism of "metal gel-shift" by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally used method for determining approximate molecular weight (MW) in protein research. Migration of protein that does not correlate with formula MW, termed "gel shifting" appears to be common for histidine-rich proteins but not yet studied in detail. We investigated "gel shifting" in Ni2+-binding histidine-rich Hpn protein cloned from Helicobacter pylori strain SS1. Our data demonstrate two important factors determining "gel shifting" of Hpn, polyacrylamide-gel concentration and metal binding. Higher polyacrylamide-gel concentrations resulted in faster Hpn migration. Irrespective of polyacrylamide-gel concentration, preserved Hpn-Ni2+ complex migrated faster (3-4 kDa) than apo-Hpn, phenomenon termed "metal gel-shift" demonstrating an intimate link between Ni2+ binding and "gel shifting". To examine this discrepancy, eluted samples from corresponding spots on SDS-gel were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The MW of all samples was the same (6945.66±0.34 Da) and identical to formula MW with or without added mass of Ni2+. MALDI-TOF-MS of Ni2+-treated Hpn revealed that monomer bound up to six Ni2+ ions non-cooperatively, and equilibrium between protein-metal species was reliant on Ni2+ availability. This corroborates with gradually increased heterogeneity of apo-Hpn band followed by compact "metal-gel shift" band on SDS-PAGE. In view of presented data metal-binding and "metal-gel shift" models are discussed.

Hg2+-trapping beads: Hg2+-specific recognition through thymine-Hg(II)-thymine base pairing.

Mercury pollution poses a severe threat to human health. To remove Hg(2+) from contaminated water, we synthesized Hg(2+)-trapping beads that include oligo-thymidine functionalities that can form thymine-Hg(II)-thymine base pairs on the solid support. The beads can selectively trap Hg(2+) even in the presence of other metal cations. More interestingly, Hg(2+)-trapping efficiency was higher in the presence of the co-existing cations. Thus, the developed Hg(2+)-trapping beads can capture Hg(2+) without affecting the mineral balance of water so much. The Hg(2+)-trapping beads presented here show promise for removing Hg(2+) from environmental water.

Improved protein overexpression and purification strategies for structural studies of cyanobacterial metal-responsive transcription factor, SmtB from marine Synechococcus sp. PCC 7002.

There are structural and functional differences in SmtB homologs, metal-responsive transcription factors responsible for sensing of excess heavy metal ions in marine and freshwater cyanobacterial strains. The structure of SmtB from freshwater Synechococcus sp. strain PCC 7942 is elucidated with nuclear magnetic resonance (NMR) and crystallography techniques. But knowledge about the functioning of SmtB homologs from marine species is limited till date. To enable NMR spectroscopic studies for investigating structural and functional aspects, modified protocols with higher yields of isotopically labeled SmtB, from marine species like Synechococcus sp. PCC 7002 are essential. In this study, smtB gene was cloned from genome of Synechococcus sp. PCC 7002 and overexpression protocol for recombinant SmtB was standardized in Escherichia coli containing T7 RNA polymerase/promoter system. Further, the protocol for large-scale production, isotope labeling with (15)N, and purification of recombinant SmtB in E. coli BL21(DE3)/pLysS cells was developed. Purified recombinant protein was successfully used for NMR spectroscopy experiments. These results indicate that the overexpression technique now developed is applicable to the structural and functional studies for the proteins being homologous to cyanobacterial SmtB from Synechococcus sp. PCC 7002.

MKRN expression pattern during embryonic and post-embryonic organogenesis in rice (Oryza sativa L. var. Nipponbare).

Rice MKRN is a member of the makorin RING finger protein gene (MKRN) family, which encodes a protein with a characteristic array of zinc-finger motifs conserved in various eukaryotes. Using non-radioactive in situ hybridization, we investigated the spatio-temporal gene expression pattern of rice MKRN during embryogenesis, imbibition, seminal and lateral root development of Oryza sativa L. var. Nipponbare. MKRN expression was ubiquitous during early organogenesis in the embryo along the apical-basal and radial axes. The expression of MKRN decreased during embryonic organ elongation and maturation compared to early embryogenesis, but increased again during imbibition. Tissue-specific and position-dependent MKRN expression was found during embryonic and post-embryonic root and shoot development. Meristematic cells ubiquitously expressed MKRN transcripts, while differentiating cells showed a gradual reduction and termination of MKRN expression. Interestingly, during post-germination MKRN expression was prominent and continued in the metabolically active, differentiated companion cells of the phloem. The differential expression pattern was observed both in the differentiating and differentiated cells. Also, MKRN was expressed in the various developmental stages of the lateral root primordia and the cells surrounding them. Expression of MKRN was also observed after periclinal division of the presumptive pericycle founder cells. The MKRN expression pattern during development of various growth stages suggests an important role of makorin RING finger protein gene (MKRN) in embryonic and post-embryonic organogenesis in both apical-basal and radial developmental axes of rice.

Elongation factor G is a critical target during oxidative damage to the translation system of Escherichia coli.

Elongation factor G (EF-G), a key protein in translational elongation, is known to be particularly susceptible to oxidation in Escherichia coli. However, neither the mechanism of the oxidation of EF-G nor the influence of its oxidation on translation is fully understood. In the present study, we investigated the effects of oxidants on the chemical properties and function of EF-G using a translation system in vitro derived from E. coli. Treatment of EF-G with 0.5 mM H(2)O(2) resulted in the complete loss of translational activity. The inactivation of EF-G by H(2)O(2) was attributable to the oxidation of two specific cysteine residues, namely, Cys(114) and Cys(266), and subsequent formation of an intramolecular disulfide bond. Replacement of Cys(114) by serine rendered EF-G insensitive to oxidation and inactivation by H(2)O(2). Furthermore, generation of the translation system in vitro with the mutated EF-G protected the entire translation system from oxidation, suggesting that EF-G might be a primary target of oxidation within the translation system. Oxidized EF-G was reactivated via reduction of the disulfide bond by thioredoxin, a ubiquitous protein that mediates dithiol-disulfide exchange. Our observations indicate that the translational machinery in E. coli is regulated, in part, by the redox state of EF-G, which might depend on the balance between the supply of reducing power and the degree of oxidative stress.

A molecular insight into Darwin's "plant brain hypothesis" through expression pattern study of the MKRN gene in plant embryo compared with mouse embryo.

MKRN gene family encodes zinc ring finger proteins characterized by a unique array of motifs (C3H, RING and a characteristic cys-his motif) in eukaryotes. To elucidate the function of the MKRN gene and to draw an analogy between plant root apical meristem and animal brain, we compared the gene expression pattern of MKRN in plant seeds with that of mouse embryo. The spatio-temporal expression of MKRN in seeds of pea and rice was performed using non radioactive mRNA in situ hybridization (NRISH) with DIG and BIOTIN labeled probes for pea and rice embryos respectively. Images of MKRN1 expression in e10.5 whole mount mouse embryo, hybridized with DIG labeled probes, were obtained from the Mouse Genome Database (MGD). MKRN transcripts were expressed in the vascular bundle, root apical meristem (RAM) and shoot apical meristem (SAM) in pea and rice embryos. The spatial annotation of the MKRN1 NRISH of whole mount mouse embryo shows prominent localization of MKRN1 in the brain, and its possible expression in spinal cord and the genital ridge. Localization of MKRN in the anterior and posterior ends of pea and rice embryo suggests to the probable role it may have in sculpting the pea and rice plants. The expression of MKRN in RAM may give a molecular insight into the hypothesis that plants have their brains seated in the root. The expression of MKRN is similar in functionally and anatomically analogous regions of plant and animal embryos, including the vascular bundle (spinal cord), the RAM (brain), and SAM (genital ridge) thus paving way for further inter-kingdom comparison studies.

Comparative study of the different mechanisms for zinc ion stress sensing in two cyanobacterial strains, Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803.

In response to an increased level of Zn(2+), Synechococcus sp. PCC 7942 expresses SmtA, a metallothionein-like metal-chelating protein, while Synechocystis sp. PCC 6803 expresses ZiaA, a transporter of Zn(2+). The gene expression of these proteins is regulated by repressor protein, SmtB and ZiaR, respectively. In spite of contributing to different response systems, both repressor proteins belong to the ArsR family and are highly homologous to each other. To understand the different systems responsible for dealing with excess Zn(2+), we examined the cis-elements in the promoter regions of smtA and ziaA, as well as the binding affinities of recombinant SmtB and ZiaR proteins. The operator/promoter region of smtA included two palindromic sequences and that of ziaA included one. Electrophoretic mobility shift assay revealed that SmtB formed four different complexes with the operator/promoter region of smtA, whereas it formed only two different complexes with the corresponding region of ziaA. For ZiaR, the corresponding results were quite the same as those for SmtB. Furthermore, the complex formation between SmtB and operator/promoter regions is inhibited in the presence of Zn(2+) at higher concentrations than 16 µM. On the other hand, the corresponding Zn(2+) concentration is 128 µM. These results demonstrate that the degrees of protein-DNA complex formation between repressor proteins and the operator/promoter regions of regulated genes depend on the structures of the operator/promoter regions, and the effects of Zn(2+) on the dissociation of these complexes are mainly associated with the structures of the repressors.

Immunohistochemical localization of apyrase during initial differentiation and germination of pea seeds.

Localization of the 49-kDa apyrase (ATP diphosphohydrolase, EC3.6.1.5; DDBJ/EMBL/GenBank BAB40230) was investigated during early stages of germination of pea (Pisum sativum L. var. Alaska) at the organ, tissue, cellular, and sub-cellular level using light-microscopical immunohistochemistry. Whole mount tissues were immuno-reacted with anti-APY1 serum, pre-immune serum or anti-actin antibody for control. Antigen to the anti-APY1 serum was not detected until 16 h after sowing (26 h after start of imbibition), when the antigen was detected throughout the tissue, especially in the epidermis and cortex. At 35 h after sowing, the younger regions including the root tip and the tip of the stele were more strongly stained than the control. Both, epidermal and cortical cells of the epicotyl and root tip were stained. The stain was mainly localized in the cytoplasm and around nuclei in the apical meristem and the root tip, while vacuoles and cell walls were not stained. At 62 h, there was major staining in the plumule, hook, and elongating regions of the epicotyl and in the region between cotyledons and the epicotyl. After 84 h, lateral root primordia were stained. The pre-immune serum showed virtually no staining while the anti-actin antibody reacted solely with the cytoplasm. Since the antigen to the anti-APY1 serum was primarily found in the cytoplasm and around nuclei in elongating and differentiating tissues and labeling declined in mature tissues, it is suggested that apyrases may play a role in growth and development of tissues, for example, lateral roots.

NMR-based characterization of a refolding intermediate of beta2-microglobulin labeled using a wheat germ cell-free system.

In patients with dialysis-related amyloidosis, beta2-microglobulin (beta2-m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of beta2-m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non-native trans-Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell-free protein synthesis and NMR techniques. The HSQC spectra of beta2-ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the beta-sheet regions especially the last half of the betaB strand and the first half of the betaE strand, both suggested to be important for amyloidogenicity, may transform beta2-m into an amyloidogenic structure.

TTP at Ser245 phosphorylation by AKT is required for binding to 14-3-3.

Transferrin receptor trafficking protein (TTP) is a key molecule for selective internalization of the transferrin receptor (Tf-R) through endocytic protein complexes. To identify the proteins that directly regulate TTP, we performed a yeast two-hybrid analysis and identified 14-3-3, which can modulate the activation state of target proteins. Subsequent analyses demonstrated that TTP directly binds to multiple 14-3-3 isotypes via its Ser(245) residue (Ser(246) in human) and that these proteins are associated at the plasma membrane. Ser(245) was also found to be a substrate for AKT and the resulting Ser(245) phosphorylation induced the TTP-14-3-3 interaction. Exposure to hydrogen peroxide rapidly enhanced this association in an ovarian cell line. These results suggest that TTP Ser(245) is the principal target for the modulation of this protein via the AKT signalling cascade.

Sequence, expression and tissue localization of a gene encoding a makorin RING zinc-finger protein in germinating rice (Oryza sativa L. ssp. Japonica) seeds.

The makorin (MKRN) RING finger protein gene family encodes proteins (makorins) with a characteristic array of zinc-finger motifs and which are present in a wide array of eukaryotes. In the present study, we analyzed the structure and expression of a putative makorin RING finger protein gene in rice (Oryza sativa L. ssp. Japonica cv. Nipponbare). From the analysis of the genomic (AP003543), mRNA (AK120250) and deduced protein (BAD61603) sequences of the putative MKRN gene of rice, obtained from GenBank, we found that it was indeed a bona fide member of the MKRN gene family. The rice MKRN cDNA encoded a protein with four C3H zinc-finger-motifs, one putative Cys-His zinc-finger motif, and one RING zinc-finger motif. The presence of this distinct motif organization and overall amino acid identity clearly indicate that this gene is indeed a true MKRN ortholog. We isolated RNA from embryonic axes of rice seeds at various stages of imbibition and germination and studied the temporal expression profile of MKRN by RT-PCR. This analysis revealed that MKRN transcripts were present at all the time points studied. It was at very low levels in dry seeds, increased slowly during imbibition and germination, and slightly declined in the seedling growth stage. After 6days of germination, an organ-dependent expression pattern of MKRN was observed: highest in roots and moderate in leaves. Similarly to MKRN transcripts, transcripts of cytoskeletal actin and tubulin were also detected in dry embryos, steadily increased during imbibition and germination and leveled off after 24h of germination. We studied the spatial expression profile of MKRN in rice tissues, by using a relatively fast, simple and effective non-radioactive mRNA in situ hybridization (NRISH) technique, which provided the first spatial experimental data that hints at the function of a plant makorin. This analysis revealed that MKRN transcripts were expressed in young plumules, lateral root primordia, leaf primordia, leaves and root tissues at many different stages of germination. The presence of MKRN transcripts in dry seeds, its early induction during germination and its continued spatiotemporal expression during early vegetative growth suggest that MKRN has an important role in germination, leaf and lateral root morphogenesis and overall development in rice.

A novel way to express proline-selectively labeled proteins with a wheat germ cell-free protein synthesis system.

For high-throughput protein structural analyses, it is essential to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones involving Escherichia coli cells, have been developed, the number of overexpressed proteins exhibiting the same biological activities as those of the native ones is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the synthesized proteins that should function in solution were found to be in soluble forms. This suggests the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we developed a selective labeling technique for amino acids having amide functional groups (other than proline residues) involving the use of several inhibitors for transaminases. This paper in turn describes a proline-selective labeling technique. Based on our results, we have succeeded in constructing a complete amino acid selective labeling technique for the wheat germ cell-free protein synthesis system.

A novel way of amino acid-specific assignment in (1)H-(15)N HSQC spectra with a wheat germ cell-free protein synthesis system.

For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized (15)N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the (1)H-(15)N HSQC spectra for native proteins and the corresponding ones for synthesized ones. In this study, we developed a convenient and reliable method for amino acid selective assignment in (1)H-(15)N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in (1)H-(15)N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.

Studies on the protein-DNA complex formation between the cyanobacterial transcription factors, SmtB and its homologues, functioning as zinc-ion sensors and the recognition DNA sequences.

In cyanobacterium Synechococcus sp. PCC 7942, SmtB, functioning as the sensor to heavy-metal ions (notably Zn-ion) in the dimer form, represses the transcription of smtA gene encoding metallothionein-like protein. There are two recognition DNA sequences in the operator/promoter region of smtA gene, and the binding of SmtB with Zn-ions reduces the affinities to these sequences. This induces the activation of smtA transcription. In this study, we have studied the functional differences between these two recognition DNA sequences, with spectroscopic (hetero-nuclear NMR and UV-resonance Raman) and biochemical methods. On the basis of the results obtained here, we clarified the regulation mechanism of the smtA expression. Similar regulatory system for the tolerance to Zn-ion stress is found in another cyanobacterium. We also found that the functionally important differences between these two systems are mainly due to the structural differences of recognition DNA sequences.

Gene structure and transcriptional regulation specific to the groESL operon from the psychrophilic bacterium Colwellia maris.

The groESL operon of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The operon contains two ORFs of 291 bp and 1,650 bp separated by 210 bp. Northern blot analysis suggested that the groESL operon was transcribed as a bicistronic mRNA, and that the amount of mRNA markedly increased after the temperature was raised from 10 degrees C to 20 degrees C. Although the optimum temperatures for GroESL function are different in psychrophilic, mesophilic, and thermophilic bacteria, the deduced amino acid sequences of C. maris GroES and GroEL showed remarkably high similarity with those of GroES and GroEL from mesophilic and thermophilic bacteria. A putative promoter similar to the Escherichia coli sigma(32) consensus sequence was identified. One specific feature of C. maris groESL was that in the putative untranslated region the G+C content was about 24 mol%, which is much lower than that of mesophilic bacteria such as E. coli. The low G+C content may be important for maintaining transcription at low temperatures.

A wheat germ cell-free system is a novel way to screen protein folding and function.

For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two (15)N-labeled proteins and obtained (1)H-(15)N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and (1)H-(15)N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell-free protein synthesis system to high-throughput protein structural analysis.

NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif.

Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.