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Antimicrobial peptides (AMPs) have been considered as the alternatives to antibiotics because of their less susceptibility to microbial resistance. However, compared with conventional antibiotics they show relatively low activity and the consequent high cost and nonspecific cytotoxicity, hindering their clinical application. What’s more, engineering of AMPs is a great challenge due to the inherent complexity in their sequence, structure, and function relationships. Here, we report an evolution-based strategy for improving the antifungal activity of a nematode-sourced defensin (Cremycin-5). This strategy utilizes a sequence-activity comparison between Cremycin-5 and its functionally diverged paralogs to identify sites associated with antifungal activity for screening of enhanceable activity-modulating sites for subsequent saturation mutagenesis. Using this strategy, we identified a site (Glu-15) whose mutations with nearly all other types of amino acids resulted in a universally enhanced activity against multiple fungal species, which is thereby defined as a Universally Enhanceable Activity-Modulating Site (UEAMS). Especially, Glu15Lys even exhibited >9-fold increased fungicidal potency against several clinical isolates of Candida albicans through inhibiting cytokinesis. This mutant showed high thermal and serum stability and quicker killing kinetics than clotrimazole without detectable hemolysis. Molecular dynamic simulations suggest that the mutations at the UEAMS likely limit the conformational flexibility of a distant functional residue via allostery, enabling a better peptide–fungus interaction. Further sequence, structural, and mutational analyses of the Cremycin-5 ortholog uncover an epistatic interaction between the UEAMS and another site that may constrain its evolution. Our work lights one new road to success of engineering AMP drug leads.

Benzopyrylium salts are an unexplored class of compounds and as a first, this study reports them as potential therapeutic agents. In this effort we pursue the synthesis and in vitro anticancer, antibacterial and antioxidant properties of some novel benzopyrylium salts. The benzopyrylium salts were synthesized and further characterized via UV-vis, IR, (1)H-NMR, (13)C-NMR and mass spectrometry. The benzopyrylium salts were tested in vitro for anticancer activity across NCI 60 cell line panel. PS-CP-4MO showed the best activity against the MDA-MB-435 cell line of melanoma cancer in terms of the least GI(50) (1.78 μM), TGI (3.47 μM) and LC(50) (6.77 μM) values and showed selectivity against melanoma, colon cancer and leukemia. Mechanistic studies indicate that this compound inhibits MCF-7 cancer cells by inducing apoptosis and abrogates colony formation and wound healing in the cancer cells. Antibacterial studies show that some of the benzopyrylium salts are active on S. aureus (ATCC 29213) and the best active compound PS-CP-5Cl has a MIC of 8 μg/mL. Antioxidant studies indicate that they have good free radical scavenging properties (PS-CP-5Cl showed activity 1.48 times ascorbic acid). Fulfillment of the Lipinski’s parameters of the benzopyrylium salts in silico showed tremendous drug likeness as potential pharmacophore leads.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spread worldwide in 2020, is an urgent problem to be overcome. The ORF8 of SARS-CoV-2 has been suggested to be associated with the symptoms of COVID-19, according to reports of clinical studies. However, little is known about the function of ORF8. As one of the ways to advance the functional analysis of ORF8, mass production of ORF8 with the correct three-dimensional structure is necessary. In this study, we attempted to produce ORF8 protein by chemically inducible protein production system using tobacco BY-2 cells. An ORF8-producing line was generated by the Agrobacterium method. As a result, the production of ORF8 of 8.8 ± 1.4 mg/L of culture medium was confirmed. SDS-PAGE and nuclear magnetic resonance (NMR) analysis confirmed that the ORF8 produced by this system is a dimeric form with a single structure, unlike that produced in Escherichia coli. Furthermore, it was suggested that the ORF8 produced by this system was glycosylated. Through this study, we succeeded in producing ORF8 folded into a single structure in a chemically inducible protein production system using tobacco BY-2 cells. It is expected that the functional analysis of ORF8 will be advanced using the ORF8 produced by this system and that it will greatly contribute to the development of antibodies and therapeutic agents targeting ORF8.

The development of eukaryote-derived antimicrobial peptides as systemically administered drugs has proven a challenging task. Here, we report the first human oral actinomyces-sourced defensin-actinomycesin-that shows promise for systemic therapy. Actinomycesin and its homologs are only present in actinobacteria and myxobacteria, and share similarity with a group of ancient invertebrate-type defensins reported in fungi and invertebrates. Signatures of natural selection were detected in defensins from the actinomyces colonized in human oral cavity and ruminant rumen and dental plaque, highlighting their role in adaptation to complex multispecies bacterial communities. Consistently, actinomycesin exhibited potent antibacterial activity against oral bacteria and clinical isolates of Staphylococcus and synergized with two classes of human salivary antibacterial factors. Actinomycesin specifically inhibited bacterial peptidoglycan synthesis and displayed weak immunomodulatory activity and low toxicity on human and mammalian cells and ion channels in the heart and central nervous system. Actinomycesin was highly efficient in mice infected with Streptococcus pneumoniae and mice with MRSA-induced experimental peritoneal infection. This work identifies human oral bacteria as a new source of systemic anti-infective drugs. ispartof: EMBO MOLECULAR MEDICINE vol:14 issue:2 ispartof: location:England status: published

Betalain pigments are mainly produced by plants belonging to the order of Caryophyllales. Betalains exhibit strong antioxidant activity and responds to environmental stimuli and stress in plants. Recent reports of antioxidant, anti-inflammatory and anti-cancer properties of betalain pigments have piqued interest in understanding their biological functions. We investigated the effects of betalain pigments (betanin and isobetanin) derived from red-beet on amyloid-β (Aβ) aggregation, which causes Alzheimer's disease. Non-specific inhibition of Aβ aggregation against Aβ40 and Aβ42 by red-beet betalain pigments, in vitro was demonstrated using the thioflavin t fluorescence assay, circular dichroism spectroscopy analysis, transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, we examined the ability of red-beet betalain pigments to interfere with Aβ toxicity by using the transgenic Caenorhabditis elegans model, which expresses the human Aβ42 protein intracellularly within the body wall muscle. It responds to Aβ-toxicity with paralysis and treatment with 50 μM red-beet betalain pigments significantly delayed the paralysis of C. elegans. These results suggest that betalain pigments reduce Aβ-induced toxicity.

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five β-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel β-barrel structure. However, the β-strands were found to display a unique topology, one pair of these β-strands formed a parallel β-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern–triggered immunity in N. benthamiana.

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 β-KTx-like two-domain structure, in which the N- and C-terminal domains form a random coil and an α-β-β 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.

Functional screening of effector candidates using a transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both the generation of reactive oxygen species (ROS) triggered by the bacterial pathogen-associated molecular pattern (PAMP), flg22, and the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five β-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel β-barrel structure. However, the β-strands were found to display a unique topology, one pair of these β-strands formed a parallel β-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress PAMP-triggered immunity (PTI) in N. benthamiana.

Nuclear magnetic resonance (NMR) data directly indicated a Ca2+-dependent interaction between calmodulin (CaM) and CoDN3, a small effector of the plant pathogenic fungus Colletotrichum orbiculare, which is the causal agent of cucumber anthracnose. The overall conformation of CoDN3 is intrinsically disordered, and the CaM-binding site spans residues 34–53 of its C-terminal region. Experiments employing a chemically synthesized peptide corresponding to the CaM-binding site indicated that the CaM-binding region of CoDN3 in the Ca2+-bound CaM complex takes an α-helical conformation. Cell death suppression assay using a CoDN3 mutant lacking the CaM-binding ability suggested that the wild type CaM-binding site is necessary for full CoDN3 function in vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has the characteristic accessory protein ORF8. Although clinical reports indicate that ORF8 variant strains (Δ382 and L84S variants) are less likely to cause severe illness, functional differences between wild-type and variant ORF8 are unknown. Furthermore, the physicochemical properties of the ORF8 protein have not been analyzed. In this study, the physicochemical properties of the wild-type ORF8 and its L84S variant were analyzed and compared. Using the tobacco BY-2 cell production system, which has been successfully used to produce the wild-type ORF8 protein with a single conformation, was used to successfully produce the ORF8 L84S variant protein at the same level as wild-type ORF8. The produced proteins were purified, and their temperature and pH dependencies were examined using nuclear magnetic resonance spectra. Our data suggested that the wild-type and L84S variant ORF8 structures are highly stable over a wide temperature range. Both proteins displayed an aggregated conformation at higher temperature that reverted when the temperature was decreased to room temperature. Moreover, ORF8 precipitated at acidic pH and this precipitation was reversed when the solution pH was shifted to neutral. Interestingly, the L84S variant exhibited greater solubility than wild-type ORF8 under acidic conditions. Thus, the finding indicated that conformational stability and reversibility of ORF8 are key properties related to function in oppressive environments.

A model legume, Medicago truncatula, has over 600 nodule-specific cysteine-rich (NCR) peptides required for symbiosis with rhizobia. Among them, NCR169, an essential factor for establishing symbiosis, has four cysteine residues that are indispensable for its function. However, knowledge of NCR169 structure and mechanism of action is still lacking. In this study, we solved two NMR structures of NCR169 caused by different disulfide linkage patterns. We show that both structures have a consensus C-terminal β-sheet attached to an extended N-terminal region with dissimilar features; one moves widely, whereas the other is relatively stapled. We further revealed that the disulfide bonds of NCR169 contribute to its structural stability and solubility. Regarding the function, one of the NCR169 oxidized forms could bind to negatively charged bacterial phospholipids. Furthermore, the positively charged lysine-rich region of NCR169 may be responsible for its antimicrobial activity against Escherichia coli and Sinorhizobium meliloti. This active region was disordered even in the phospholipid bound state, suggesting that the disordered conformation of this region is key to its function. Morphological observations suggested the mechanism of action of NCR169 on bacteria. The present study on NCR169 provides new insights into the structure and function of NCR peptides.

In quinoa seedlings, the pigment betalain accumulates in the hypocotyl. To isolate the genes involved in betalain biosynthesis in the hypocotyl, we performed ethyl methanesulfonate (EMS) mutagenesis on the CQ127 variety of quinoa seedlings. While putative amaranthin and celosianin II primarily accumulate in the hypocotyls, this process produced a green hypocotyl mutant (ghy). This MutMap+ method using the quinoa draft genome revealed that the causative gene of the mutant is CqCYP76AD1-1. Our results indicated that the expression of CqCYP76AD1-1 was light-dependent. In addition, the transient expression of CqCYP76AD1-1 in Nicotiana benthamiana leaves resulted in the accumulation of betanin but not isobetanin, and the presence of a polymorphism in CqCYP76A1-2 in the CQ127 variety was shown to have resulted in its loss of function. These findings suggested that CqCYP76AD1-1 is involved in betalain biosynthesis during the hypocotyl pigmentation process in quinoa. To our knowledge, CqCYP76AD1-1 is the first quinoa gene identified by EMS mutagenesis using a draft gene sequence.

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 β-toxin acting on K+-channels (β-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.

In our previous study, functional ability and conformational stability had been examined for C-terminal deletion mutants of a 148-residue Ca2+-binding protein, chicken calmodulin (CaM). In that study, we had reported that a mutant named CCMΔ5, missing five residues at the C-terminus, activates CaM-target as much as full-length CaM does. This finding is intriguing because CCMΔ5 lacks the key residues, Met144 and Met145, for the target activation. To uncover why CCMΔ5 displays proper function, here we report the three-dimensional structure of CCMΔ5 bound to the peptide derived from skeletal muscle myosin light-chain kinase (skMLCK). The structure determination was achieved using multidimensional nuclear magnetic resonance (NMR) spectroscopy. The complex structure of CCM∆5-skMLCK was compared to that of wild CaM-skMLCK. The results showed that the orientation of helix-1 and helix-5 in CCM∆5 differs from those in wild CaM. Moreover, distinctive hydrophobic interaction manner was found in the binding between CCM∆5 and peptide; Phe141, Ala128, Met109, Leu105 and Phe92 of CCM∆5 contribute to the interaction with Trp4 of the skMLCK peptide. Abbreviations: CaM, calmodulin; CCMΔX, a deletion mutant of CaM that lacks X C-terminal residues; NMR, Nuclear magnetic resonance; PDB, Protein date bank; skMLCK, skeletal muscle myosin light-chain kinase; TOF-MS, Time-of-flight mass spectrometry; RMSD, root mean square deviation; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis Citation: Vu Van Dung, Umetsu Y., Ohki S., 2017. Three-dimensional structure of the complex between calmodulin mutant lacking the c-terminal five residues and the calmodulin-binding peptide derived from skeletal muscle myosin light-chain kinase. Tap chi Sinh hoc, 39(3): 309-319. DOI: 10.15625/0866-7160/v39n3.10111. *Corresponding author: vandungbio46@yahoo.com Received 19 June 2017, accepted 20 August 2017

Two mGluR7-derived peptides corresponding to residues 856 to 879 and 856 to 875 are known to bind to Ca2+-saturated calmodulin (Ca2+/CaM), and their binding manners are thought to differ. Met872 function is believed as one of the anchor residues for CaM-binding only in the shorter peptide. To uncover the role of Met872 in CaM-binding, we prepared a mutant of the long peptide, mGluR7 (M872A), in which Met872 was replaced with Ala. We used the mutant together with the two peptides to perform NMR-titration experiments to monitor interaction with stable isotope-labeled CaM. Interaction of Ca2+/CaM with mGluR7 (M872A) caused a spectrum that differed from that of Ca2+/CaM with the long peptide, suggesting that Met872 of mGluR7 could be involved in CaM-binding even in the long peptide. Analyses of all NMR data suggested that the binding between Ca2+/CaM and mGluR7 occurs in some conformational equilibrium manner. The unique CaM-binding properties caused by Met872 may be related to mGluR7's function.

Halophytes are plants that grow in high-salt environments and form characteristic epidermal bladder cells (EBCs) that are important for saline tolerance. To date, however, little has been revealed about the formation of these structures. To determine the genetic basis for their formation, we applied ethylmethanesulfonate mutagenesis and obtained two mutants with reduced levels of EBCs (rebc) and abnormal chloroplasts. In silico subtraction experiments revealed that the rebc phenotype was caused by mutation of REBC, which encodes a WD40 protein that localizes to the nucleus and chloroplasts. Phylogenetic and transformant analyses revealed that the REBC protein differs from TTG1, a WD40 protein involved in trichome formation. Furthermore, rebc mutants displayed damage to their shoot apices under abiotic stress, suggesting that EBCs may protect the shoot apex from such stress. These findings will help clarify the mechanisms underlying EBC formation and function., Tomohiro Imamura et al. report that a WD40 protein REBC, which differs from the trichome formation-linked WD40 protein TTG1, plays a critical role in epidermal bladder cell formation in quinoa. They further reveal a protective role of the epidermal bladder cells against the shoot apex damage by abiotic stress, especially salt stress, shedding light on the mechanisms underlying epidermal bladder cell formation and function.

Nuclear magnetic resonance (NMR) data directly indicated a Ca

Summary Betalains are plant pigments primarily produced by plants of the order Caryophyllales. Because betalain possesses anti‐inflammatory and anticancer activities, it may be useful as a pharmaceutical agent and dietary supplement. Recent studies have identified the genes involved in the betalain biosynthesis of betanin. Amaranthin and celosianin II are abundant in the quinoa (Chenopodium quinoa Willd.) hypocotyl, and amaranthin comprises glucuronic acid bound to betanin; therefore, this suggests the existence of a glucuronyltransferase involved in the synthesis of amaranthin in the quinoa hypocotyl. To identify the gene involved in amaranthin biosynthesis, we performed a BLAST analysis and phylogenetic tree analysis based on sequences homologous to flavonoid glycosyltransferase, followed by expression analysis on the quinoa hypocotyl to obtain three candidate proteins. Production of amaranthin in a transient Nicotiana benthamiana expression system was evaluated for these candidates and one was identified as having the ability to produce amaranthin. The gene encoding this protein was quinoa amaranthin synthetase 1 (CqAmaSy1). We also created a transgenic tobacco bright yellow‐2 (BY‐2) cell line wherein four betalain biosynthesis genes were introduced to facilitate amaranthin production. This transgenic cell line produced 13.67 ± 4.13 μm (mean ± SEM) amaranthin and 26.60 ± 1.53 μm betanin, whereas the production of isoamaranthin and isobetanin could not be detected. Tests confirmed the ability of amaranthin and betanin to slightly suppress cancer cell viability. Furthermore, amaranthin was shown to significantly inhibit HIV‐1 protease activity, whereas betanin did not.

Scorpion K(+) channel toxins and insect defensins share a conserved three-dimensional structure and related biological activities (defense against competitors or invasive microbes by disrupting their membrane functions), which provides an ideal system to study how functional evolution occurs in a conserved structural scaffold. Using an experimental approach, we show that the deletion of a small loop of a parasitoid venom defensin possessing the "scorpion toxin signature" (STS) can remove steric hindrance of peptide-channel interactions and result in a neurotoxin selectively inhibiting K(+) channels with high affinities. This insect defensin-derived toxin adopts a hallmark scorpion toxin fold with a common cysteine-stabilized α-helical and β-sheet motif, as determined by nuclear magnetic resonance analysis. Mutations of two key residues located in STS completely diminish or significantly decrease the affinity of the toxin on the channels, demonstrating that this toxin binds to K(+) channels in the same manner as scorpion toxins. Taken together, these results provide new structural and functional evidence supporting the predictability of toxin evolution. The experimental strategy is the first employed to establish an evolutionary relationship of two distantly related protein families. ispartof: Molecular Biology and Evolution vol:31 issue:3 pages:546-59 ispartof: location:United States status: published

Calmodulin (CaM) binds to the FERM domain of 80 kDa erythrocyte protein 4.1R (R30) independently of Ca(2+) but, paradoxically, regulates R30 binding to transmembrane proteins in a Ca(2+)-dependent manner. We have previously mapped a Ca(2+)-independent CaM-binding site, pep11 (A(264)KKLWKVCVEHHTFFR), in 4.1R FERM domain and demonstrated that CaM, when saturated by Ca(2+) (Ca(2+)/CaM), interacts simultaneously with pep11 and with Ser(185) in A(181)KKLSMYGVDLHKAKD (pep9), the binding affinity of Ca(2+)/CaM for pep9 increasing dramatically in the presence of pep11. Based on these findings, we hypothesized that pep11 induced key conformational changes in the Ca(2+)/CaM complex. By differential scanning calorimetry analysis, we established that the C-lobe of CaM was more stable when bound to pep11 either in the presence or absence of Ca(2+). Using nuclear magnetic resonance spectroscopy, we identified 8 residues in the N-lobe and 14 residues in the C-lobe of pep11 involved in interaction with CaM in both of presence and absence of Ca(2+). Lastly, Kratky plots, generated by small-angle X-ray scattering analysis, indicated that the pep11/Ca(2+)/CaM complex adopted a relaxed globular shape. We propose that these unique properties may account in part for the previously described Ca(2+)/CaM-dependent regulation of R30 binding to membrane proteins.

We propose a novel Madake (Phyllostachys bambusoides) bamboo suspension culture model for investigation of key enzyme(s) activity involved in growth/differentiation. Sedimented Cell Volume (SCV) and fresh weight (FW) of the suspension cultured cells reached 34% (v/v) and 8.7 g in 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium while only 7% (v/v) SCV and 1.9 g FW of the cells in 10 μM gibberellic acid (GA3)-containing medium in 14 days. Proportion of mitotically active cells (S to G2/M phases) at a log phase was identified as 29.5% in the former cells with tiny cytoplasmic features while 5.4% in the latter cells with elongation, wall thickening, and lignification by using flow cytometry and laser scanning microscopic analysis. The total β-glucosidase (BGL) activity under the 2,4-D condition increased from 4.8 U in day 2 to 26.2 U in day 14 (ca. 5.5-fold) while a slight reduction, from 4.4 U in day 2 to 2.1 U in day 14 (ca. 0.5-fold), occurred when cell division was suppressed under the GA3 condition. Ratio of the BGL activity of the soluble fractions to the membrane-associated fractions varied depending of the culture condition. The ratio was stable (2 to 8) during the culture period under the 2,4-D condition. Interestingly, the activity of the soluble enzyme fractions increased up to ca. 65% under the GA3 condition in inverse proportion to the membrane-associated enzymes. All together, it was strongly suggested that the detected specificity/variability of BGL activity is potentially involved in cell division and lignification in Madake bamboo cells.

A number of multifunctional ABx-type monomers exist in plant metabolites, and studies on the formation of hyperbranching polymers from ABx-type monomers are very significant in the development of bio-related polymeric materials. We established a method for the preparation of well-definedstructures in bio-based, hyperbranched (HB) polyarylates by the copolycondensation of caffeic acid (DHCA) as an AB_2-monomer and p-coumaric acid (4HCA) as an AB-monomer, using the highly efficient catalyst Na_2HPO_4 to regulate the polymerization speed. ^1H NMR analysis revealed the time course of the formation of the hyperbranching structures. which strongly affected the glass transition and degradation temperatures, as well as the molecular weight and composition.

Calmodulin (CaM), a Ca(2+)-binding protein, is a well-known regulator of various cellular functions. One of the targets of CaM is metabotropic glutamate receptor 7 (mGluR7), which serves as a low-pass filter for glutamate in the pre-synaptic terminal to regulate neurotransmission. Surface plasmon resonance (SPR), circular dichroism (CD) spectroscopy and nuclear magnetic spectroscopy (NMR) were performed to study the structure of the peptides corresponding to the CaM-binding domain of mGluR7 and their interaction with CaM. Unlike well-known CaM-binding peptides, mGluR7 has a random coil structure even in the presence of trifluoroethanol. Moreover, NMR data suggested that the complex between Ca(2+)/CaM and the mGluR7 peptide has multiple conformations. The mGluR7 peptide has been found to interact with CaM even in the absence of Ca(2+), and the binding is directed toward the C-domain of apo-CaM rather than the N-domain. We propose a possible mechanism for the activation of mGluR7 by CaM. A pre-binding occurs between apo-CaM and mGluR7 in the resting state of cells. Then, the Ca(2+)/CaM-mGluR7 complex is formed once Ca(2+) influx occurs. The weak interaction at lower Ca(2+) concentrations is likely to bind CaM to mGluR7 for the fast complex formation in response to the elevation of Ca(2+) concentration.

Electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, flow dialysis, and bioactivity measurements were employed to investigate the roles of the C-terminal residues of calmodulin (CaM). In the present study, we prepared a series of truncated mutants of chicken CaM that lack four (CCMΔ4) to eight (CCMΔ8) residues at the C-terminal end. It was found that CCMΔ4, lacking the last four residues (M145 to K148), binds four Ca2+ ions. Further deletion gradually decreased the ability to bind the fourth Ca2+ ion, and CCMΔ8 completely lost the ability. Interestingly, both lobes of Ca2+-sturated CCMΔ5 showed instability in the conformation, although limited part in the C-lobe of Ca2+-saturated CCMΔ4 was instable. Moreover, unlike CCMΔ4, structure of the C-lobe in CCMΔ5 bound to the target displayed dissimilarity to that of CaM, suggesting that deletion of M144 changes the binding manner. Deletion of the last five residues (M144 to K148) and further truncation of the C-terminal region decreased apparent capacity for target activation. Little contribution of the last four residues including M145 was observed for structural stability, Ca2+-binding, and target activation. Although both M144 and M145 have been recognized as key residues for the function, the present data suggest that M144 is a more important residue to attain Ca2+ induced conformational change and to form a proper Ca2+-saturated conformation.

Metabotropic glutamate receptors (mGluRs) influence a variety of second-messenger systems and ion channels. The C-terminal region of group III mGluRs interacts with the Ca 2+ -binding protein calmodulin (CaM). We intend to study the interaction between Ca 2+ /CaM and the CaM-binding motifs within mGluR 7 , which is a group III mGluR. We established a recombinant protein expression and purification system for nuclear magnetic resonance (NMR) analysis of mGluR 7 peptides using Escherichia coli . Peptides of mGluR 7 conjugated to an affinity tag sequence were constructed, and protocols for expression and purification were optimized. To suppress non-specific enzymatic cleavage, the mGluR 7 fusion peptide was bound to Ca 2+ /CaM before enterokinase cleavage. This complex method for precise enzymatic reactions may be applicable for the recombinant preparation of a wide variety of peptides.

Cell motility, such as smooth muscle contraction and cell migration, is controlled by the reversible phosphorylation of the regulatory light chain of myosin II and other cytoskeletal proteins. Mounting evidence suggests that in smooth muscle cells and other types of cells in vertebrates, myosin phosphatase (MP) plays an important role in controlling the phosphorylation of myosin II as well as other cytoskeletal proteins, including ezrin, moesin, and radixin.1 MP is a holoenzyme consisting of a catalytic subunit of a type-1 Ser/Thr phosphatase (PP1C) delta isoform, a myosin phosphatase targeting subunit 1 (MYPT1), and an accessory subunit M21. In this ternary complex, MYPT1 is responsible for regulating the phosphatase activity.1 A recent X-ray crystallographic study revealed an allosteric interaction between PP1C and the N-terminal ankyrin repeat domain of MYPT1 that confers the substrate specificity of the enzyme.2 MP activity is suppressed when Thr696 or Thr853 of MYPT1 is phosphorylated by various kinases, such as ROCK, ZIPK, ILK, and PAK.1,3 However, it is still unclear how the phosphorylation of MYPT1 inhibits MP activity. The amino acid sequence around Thr696 of MYPT1 is highly conserved among MYPT1 family members including MYPT2 and MBS85. Therefore, structural insights into the inhibitory domain of MYPT1 are expected to provide new clues to fully elucidate the mechanism that controls phosphatase activity via the phosphorylation of MYPT1 or other family members involved in kinase-phosphatase crosstalk in cytoskeletal regulation. Here, we prepared a bacterial recombinant fragment of MYPT1 corresponding to residues 658 to 714, including the phosphorylation site Thr696, and determined its three-dimensional structure through the use of computer-assisted distance geometry and a simulated annealing protocol combined with stable-isotope-aided multi-dimensional NMR techniques.

We established a novel strategy for preparing uniformly stable isotope-labeled proteins by using suspension-cultured plant cells and an inducible virus vector encoding the research target. By using this new method, we demonstrated the expression of three proteins, namely, Escherichia coli dihydrofolate reductase (DHFR), chicken calmodulin (CaM), and porcine protein kinase C-dependent protein phosphatase-1 inhibitor with a molecular mass of 17-kDa (CPI-17). In addition, we successfully expressed bovine pancreatic trypsin inhibitor (BPTI), which contains three pairs of disulfide bonds, as the soluble form. In the most efficient case, as little as 50 ml culture yielded 3-4 mg ^N-labeled protein suitable for NMR experiments. The ^1H-^N HSQC spectra of all of these proteins clearly indicated that their structures were identical to those of their counterparts reported previously. Thus, the present results suggest that our novel protocol is a potential method for NMR sample preparation.

Phosphorylation of endogenous inhibitor proteins specific for type-1 Ser/Thr phosphatase (PP1) provides a mechanism for reciprocal coordination of kinase and phosphatase activities. Phosphorylation of Thr38 in the inhibitor protein CPI-17 transduces G-protein-mediated signaling into a > 1000-fold increase of inhibitory potency toward myosin phosphatase. We show here the solution NMR structure of phospho-T38-CPI-17 with r. m. s. d. of 0.36 ± 0.06 Å for the backbone secondary structure, which reveals how phosphorylation triggers a conformational change and exposes the PP1 inhibitory surface. This active conformation is stabilized by the formation of a hydrophobic core of intercalated side-chains, which is not formed in a phospho-mimetic D38 mutant form of CPI-17. Thus, the profound increase in potency of CPI-17 arises from phosphorylation, conformational change and hydrophobic stabilization of a rigid structure that poses the phosphorylated residue on the protein surface and restricts its hydrolysis by myosin phosphatase. Our results provide structural insights into transduction of kinase signals by PP1 inhibitor proteins.

The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

In order to investigate the factors responsible for protein thermostability, we performed a comparative analysis. For this study, we prepared a new dataset composed of 47 homologous pairs of thermophilic and mesophilic proteins. It is the largest comparative study dataset ever presented. The frequency and substitution preference of each amino acid type in the dataset were analyzed. Two kinds of residual structural states were considered, i.e. surface (solvent-exposed) and core (buried) regions. On the surface of thermophilic proteins, higher frequencies were observed for Arg, Glu, and Tyr. Analysis of substitution preference also suggests that these often appear by replacement of other amino acid types. The results indicate that Arg, Glu, and Tyr are suitable for location on the surface of thermophilic proteins. On the other hand, at the core of thermophilic proteins, Ala is often appeared. In addition, our t-test analysis provides the first quantitative information about trends in the frequencies and substitution preferences for Cys, Gln, Met, and Ser. The results indicate that Gln and Met on the surface and Cys and Ser in the core are disadvantageous for protein thermostability.

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.

Phospho-amino acids in proteins are directly associated with phospho-receptor proteins, including protein phosphatases. Here we produced and tested a scheme for docking together interacting phospho-proteins whose monomeric 3D structures were known. The phosphate of calyculin A, an inhibitor for protein phosphatase-1 and 2A (PP1 and PP2A), or phospho-CPI-17, a PP1-specific inhibitor protein, was docked at the active site of PP1. First, a library of 186,624 virtual complexes was generated in silico, by pivoting the phospho-ligand at the phosphorus atom by step every 5 degrees on three rotational axes. These models were then graded for probability according to atomic proximity between two molecules. The predicted structure of PP1 x calyculin A complex fitted to the crystal structure with r.m.s.d. of 0.23 A, providing a validate test of the modeling method. Modeling of PP1 x phospho-CPI-17 complex yielded one converged structure. The segment of CPI-17 around phospho-Thr38 is predicted to fit in the active site of PP1. Positive charges at Arg33/36 of CPI-17 are in close proximity to Glu274 of PP1, where the sequence is unique among Ser/Thr phosphatases. Single mutations of these residues in PP1 reduced the affinity against phospho-CPI-17. Thus, the interface of the PP1 x CPI-17 complex predicted by the phospho-pivot modeling accounts for the specificity of CPI-17 against PP1.

Direct docking of nanomolecules, such as proteins, is responsible for biological signal transduction in cells. This physiological interaction is mimicked by various biosensors in nanotechnology. In...

The structures of CPI-17 (Protein kinase-C dependent protein phosphatase-1 (PP1) inhibitor of 17 kDa) in an inactive and an active form have been determined by multidimensional NMR spectroscopy. Comparison of the two structures revealed how the molecular switch turns on at atomic resolution. Using the NMR structure of CPI-17 in the active form, the binding with catalytic domain of PP1 (PP1c) was simulated and the binding model is proposed in this report. When the phospho-Thr38 docks to the catalytic site of PP1, possible interactions for the tight binding are found; one is electrostatic interaction between a negatively charged cluster on phospho-CPI-17 and an acidic groove of PP1c, and the other is hydrophobic interaction between a hydrophobic surface area of phospho-CPI-17 and a hydrophobic groove of PP1c.

We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H–15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.

Venom-derived neurotoxins are ideal probes for the investigation of structure-function relationship of ion channels and promising scaffolds for the design of ion channel-targeted drug leads as well. The discovery of highly selective toxins against a specific channel subtype facilitates the development of drugs with reduced side effects. Here, we describe the systemic characterization of a new scorpion short-chain K(+) channel blocker from Mesobuthus martensii, termed mesomartoxin (MMTX). MMTX is synthesized as a precursor comprising a signal peptide and a mature peptide of 29 residues. Nuclear magnetic resonance analysis confirmed that recombinant MMTX adopts a typical cysteine-stabilized α-helical and β-sheet fold. Electrophysiological experiments showed that MMTX exhibits high affinity for the Drosophila Shaker K(+) channel but differential selectivity on different members of the rat voltage-gated K(+) channel (Kv) family, with nanomolar affinity (IC50=15.6 nM) for rKv1.2, micromolar affinity for rKv1.3 (IC50=12.5 μM) and no activity on rKv1.1 at >50 μM. Site-directed mutagenesis of the channel pore identified a key site located on the selectivity filter of the pore, which is directly implicated in toxin binding and controls target's selectivity of the toxin. Given a key role of Kv1.2 in epilepsy, MMTX might serve as a potential drug lead for the disease.

Flagellar dyneins are essential microtubule motors in eukaryotes, as they drive the beating motions of cilia and flagella. Unlike myosin and kinesin motors, the track binding mechanism of dyneins and the regulation between the strong and weak binding states remain obscure. Here we report the solution structure of the microtubule-binding domain of flagellar dynein-c/DHC9 (dynein-c MTBD). The structure reveals a similar overall helix-rich fold to that of the MTBD of cytoplasmic dynein (cytoplasmic MTBD), but dynein-c MTBD has an additional flap, consisting of an antiparallel b sheet. The flap is positively charged and highly flexible. Despite the structural similarity to cytoplasmic MTBD, dynein-c MTBD shows only a small change in the microtubule- binding affinity depending on the registry change of coiled coil-sliding, whereby lacks the apparent strong binding state. The surface charge distribution of dynein-c MTBD also differs from that of cytoplasmic MTBD, which suggests a difference in the microtubule-binding mechanism.

Tripeptide Maternal Support In flowering plants, fertilization involves multiple gametes. The diploid zygote, which will form the embryonic plant, is surrounded by the often triploid endosperm, which provides a supportive and nourishing function. Working in Arabidopsis , Costa et al. (p. 168 ; see the Perspective by Bayer ) identified a trio of small signaling peptides that derive from the endosperm but that regulate growth of the embryo. RNA interference was used to down-regulate expression of all three peptides. Fertilization was not affected, but seed growth was. The peptides were critical for normal development of the suspensor, which tethers and nourishes the growing embryo.

Contractility of vascular smooth muscle depends on phosphorylation of myosin light chains, and is modulated by hormonal control of myosin phosphatase activity. Signaling pathways activate kinases such as PKC or Rho-dependent kinases that phosphorylate the myosin phosphatase inhibitor protein called CPI-17. Phosphorylation of CPI-17 at Thr38 enhances its inhibitory potency 1000-fold, creating a molecular on/off switch for regulating contraction. We report the solution NMR structure of the CPI-17 inhibitory domain (residues 35–120), which retains the signature biological properties of the full-length protein. The final ensemble of 20 sets of NMR coordinates overlaid onto their mean structure with r.m.s.d. values of 0.84(±0.22) A for the backbone atoms. The protein forms a novel four-helix, V-shaped bundle comprised of a central anti-parallel helix pair (B/C helices) flanked by two large spiral loops formed by the N and C termini that are held together by another anti-parallel helix pair (A/D helices) stabilized by intercalated aromatic and aliphatic side-chains. Chemical shift perturbations indicated that phosphorylation of Thr38 induces a conformational change involving displacement of helix A, without significant movement of the other three helices. This conformational change seems to flex one arm of the molecule, thereby exposing new surfaces of the helix A and the nearby phosphorylation loop to form specific interactions with the catalytic site of the phosphatase. This phosphorylation-dependent conformational change offers new structural insights toward understanding the specificity of CPI-17 for myosin phosphatase and its function as a molecular switch.

The structure of the neuronal nitric oxide synthase inhibitory protein, PIN (protein inhibitor of nNOS), has been determined by NMR spectroscopy. Two N-terminal antiparallel α-helices pack against a four-stranded antiparallel β-sheet in the C-terminal region of the protein, forming a two-layer α/β plait. The three dimensional structure of PIN resembles the fold of the B-chain of aspartylglucosaminidase. A non-prolyl cis peptide bond was found between Pro 52 and Thr 53 of the protein. PIN has a large solvent-exposed hydrophobic surface that contains a cavity and is rimmed with positive charges. This surface may serve as the primary target-binding region for this multi-functional regulatory protein.

The binding of Mg2+ to calmodulin (CaM) and the effect of Mg2+ on the binding of Ca2+-CaM to target peptides were examined using two-dimensional nuclear magnetic resonance and fluorescence spectroscopic techniques. We found that Mg2+ preferentially binds to Ca2+-binding sites I and IV of CaM in the absence of Ca2+ and that Ca2+-binding site III displays the lowest affinity for Mg2+. In contrast to the marked structural transitions induced by Ca2+ binding, Mg2+ binding causes only localized conformational changes within the four Ca2+-binding loops of CaM. Therefore, Mg2+ does not seem to be able to cause significant structural effects required for the interaction of CaM with target proteins. The presence of excess Mg2+ (up to 10 mM) does not change the order and cooperativity of Ca2+ binding to CaM, and as expected, the structure of Ca2+-saturated CaM is not affected by the presence of Mg2+. However, we found that the binding of Ca2+-saturated CaM to target peptides is affected by Mg2+ with the binding affinity decreasing as the Mg2+ concentration increases. Three different peptides, corresponding to the CaM binding domain of skeletal muscle myosin light-chain kinase (MLCK), CaM-dependent cyclic nucleotide phosphodiesterase (PDE), and smooth muscle caldesmon (CaD), were examined and show different reductions in their affinities toward CaM. The CaM-binding affinity of the MLCK peptide in the presence of 50 mM Mg2+ is approximately 40-fold lower than that seen in the absence of Mg2+, and a similar response was observed for the PDE peptide. The affinity of the CaD peptide for CaM also shows a Mg2+ dependence, though to a much lower magnitude. The Mg2+-dependent decrease in the affinities between CaM and its target peptides is an intrinsic property of Mg2+ rather than a nonspecific ionic effect, as other metal ions such as Na+ do not completely replicate the effect of Mg2+. The inhibitory effect of Mg2+ on the formation of complexes between CaM and its targets may contribute to the specificity of CaM in target activation in response to cellular Ca2+ concentration fluctuations.

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca(2+)-saturated calmodulin (Ca(2+)/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin-radixin-moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca(2+)/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca(2+)/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca(2+)/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S(71)RGISRFIPPWLKKQKS peptide (pepG) mediates Ca(2+)/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca(2+)/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca(2+)/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.