31P-nuclear magnetic resonance studies of intact plasmodia of Physarum polycephalum

Abstract31P-nuclear magnetic resonance spectra were obtained from intact plasmodial cells of Physarum polycephalum, where cytoplasmic streaming is generated by actin-myosin-ATP interaction. Several peaks were resolved and identified. They included ATP, ADP, orthophosphate and polyphosphates. Peaks for phosphocreatine, phosphoarginine or AMP were not detected. The intracellular pH and concentrations of ATP and free Mg2+ were estimated to be pH 6.9, 0.2–0.5 mM, and about 1 mM, respectively

NMR spectra of PB2 627, the RNA-binding domain in influenza A virus RNA polymerase that contains the pathogenicity factor lysine 627, and improvement of the spectra by small osmolytes

The influenza A virus, which has an RNA genome, requires RNA-dependent RNA polymerase for transcription and replication. The polymerase is comprised of the subunits PA, PB1, and PB2. The C-terminal RNA-binding domain in PB2 contains lysine 627 (PB2 627), which is associated with pathogenicity and host range. However, the structure and molecular mechanism of PB2 627 in solution remain obscure. Here, we investigated PB2 627 in solution by nuclear magnetic resonance (NMR) and detected inhomogeneity in the intensities of backbone amide proton signals due to local fluctuations in structure. To characterize the effects of chemical chaperones on spectral data and improve the data quality, we tested 20 different additives, including L-arginine L-glutamate salt, (L-arginine)2SO4, glycerol, β-octylglucoside, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Na2SO4, 1,5-diaminopentane, 1,4-diaminobutane, trehalose, sucrose, glycine, trimethylamine N-oxide, β-alanine, L-α-alanine, hydroxyectoine, betaine, L-proline, and non-detergent sulfobetaine 195, 201, and 256. We evaluated the quality of the resulting spectra by calculating the standard deviation and average of the ratio of signal intensities to noise level of amide peaks, as well as the ratio of the standard deviation to the average. NMR-profile analysis revealed diverse effects of additives on the dynamic properties of PB2 627. Based on such criteria, we found that small osmolytes such as glycine and L-α-alanine reduced structural fluctuations and improved the quality of spectral data, which is likely to facilitate a detailed NMR-based structural analysis. The methodology developed here may also be more generally useful for evaluating the effects of chemical chaperones on the structural integrity of proteins

The Arginine Residue within the C-Terminal Active Core of Bombyx mori Pheromone Biosynthesis-Activating Neuropeptide is Essential for Receptor Binding and Activation

In most lepidopteran insects, the biosynthesis of sex pheromones is regulated by pheromone biosynthesis-activating neuropeptide (PBAN). Bombyx mori PBAN (BomPBAN) consists of 33 amino acid residues and contains a C-terminus FSPRLamide motif as the active core. Among neuropeptides containing the FXPRLamide motif, the arginine (Arg, R) residue at the second position from the C-terminus is highly conserved across several neuropeptides, which can be designated as RXamide peptides. The purpose of this study was to clarify the role of the Arg residue in the BomPBAN active core. We synthesized 10-residue peptides corresponding to the C-terminal part of BomPBAN with a series of replacements at the second position from the C-terminus, termed the C2 position, and measured their efficacy in stimulating Ca2+ influx in insect cells expressing a fluorescent PBAN receptor chimera (PBANR–EGFP) using the fluorescent Ca2+ indicator, Fura Red–AM. The PBAN analogs with the C2 position replaced with alanine (Ala, A), aspartic acid (Asp, D), serine (Ser, S), or l-2-aminooctanoic acid (Aoc) decreased PBAN-like activity. RC2A (SKTRYFSPALamide) and RC2D (SKTRYFSPDLamide) had the lowest activity and could not inhibit the activity of PBAN C10 (SKTRYFSPRLamide). We also prepared Rhodamine Red-labeled peptides of the PBAN analogs and examined their ability to bind PBANR. In contrast to Rhodamine Red-PBAN C10 at 100 nM, none of the synthetic analogs exhibited PBANR binding at the same concentration. Taken together, our results demonstrate that the C2 Arg residue in BomPBAN is essential for PBANR binding and activation

Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Erratum

An erratum to the paper by Miyakawa et al. [(2007), Acta Cryst. F63, 737–739]

Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5′-GTAC and leave a 3′-TA overhang (5′-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure

Re-Evaluation of the PBAN Receptor Molecule: Characterization of PBANR Variants Expressed in the Pheromone Glands of Moths

Sex pheromone production in most moths is initiated following pheromone biosynthesis activating neuropeptide receptor (PBANR) activation. PBANR was initially cloned from pheromone glands (PGs) of Helicoverpa zea and Bombyx mori. The B. mori PBANR is characterized by a relatively long C-terminus that is essential for ligand-induced internalization, whereas the H. zea PBANR has a shorter C-terminus that lacks features present in the B. mori PBANR critical for internalization. Multiple PBANRs have been reported to be concurrently expressed in the larval CNS of Heliothis virescens. In the current study, we sought to examine the prevalence of multiple PBANRs in the PGs of three moths and to ascertain their potential functional relevance. Multiple PBANR variants (As, A, B, and C) were cloned from the PGs of all species examined with PBANR-C the most highly expressed. Alternative splicing of the C-terminal coding sequence of the PBAN gene gives rise to the variants, which are distinguishable only by the length and composition of their respective C-terminal tails. Transient expression of fluorescent PBANR chimeras in insect cells revealed that PBANR-B and PBANR-C localized exclusively to the cell surface while PBANR-As and PBANR-A exhibited varying degrees of cytosolic localization. Similarly, only the PBANR-B and PBANR-C variants underwent ligand-induced internalization. Taken together, our results suggest that PBANR-C is the principal receptor molecule involved in PBAN signaling regardless of moth species. The high GC content of the C-terminal coding sequence in the B and C variants, which makes amplification using conventional polymerases difficult, likely accounts for previous “preferential” amplification of PBANR-A like receptors from other species

Establishment of Sf9 Transformants Constitutively Expressing PBAN Receptor Variants: Application to Functional Evaluation

To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines constitutively expressing green fluorescent protein chimeras of the recently identified Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants. Fluorescent chimeras included the BommoPBANR-A, -B, and -C variants and the PsesePBANR-B and -C variants. Cell lines expressing non-chimeric BommoPBANR-B and -C variants were also generated. Functional evaluation of these transformed cell lines using confocal laser microscopy revealed that a Rhodamine Red-labeled PBAN derivative (RR-C10PBANR2K) specifically co-localized with all of the respective PBANR variants at the plasma membrane. Near complete internalization of the fluorescent RR-C10PBANR2K ligand 30 min after binding was observed in all cell lines except those expressing the BommoPBANR-A variant, in which the ligand/receptor complex remained at the plasma membrane. Fluorescent Ca2+ imaging further showed that the BommoPBANR-A cell line exhibited drastically different Ca2+ mobilization kinetics at a number of RR-C10PBANR2K concentrations including 10 μM. These observations demonstrate a clear functional difference between the BommoPBANR-A variant and the BommoPBANR-B and -C variants in terms of receptor regulation and activation of downstream effector molecules. We also found that, contrary to previous reports, ligand-induced internalization of BommoPBANR-B and BommoPBANR-C in cell lines stably expressing these variants occurred in the absence of extracellular Ca2+

Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6

AbstractKinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Alcaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR

Crystal structure of a Ca2+-dependent regulator of flagellar motility reveals the open-closed structural transition

Sperm chemotaxis toward a chemoattractant is very important for the success of fertilization. Calaxin, a member of the neuronal calcium sensor protein family, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis of ascidian, Ciona intestinalis. Here, we present the crystal structures of calaxin both in the open and closed states upon Ca2+ and Mg2+ binding. The crystal structures revealed that three of the four EF-hands of a calaxin molecule bound Ca2+ ions and that EF2 and EF3 played a critical role in the conformational transition between the open and closed states. The rotation of α7 and α8 helices induces a significant conformational change of a part of the α10 helix into the loop. The structural differences between the Ca2+- and Mg2+-bound forms indicates that EF3 in the closed state has a lower affinity for Mg2+, suggesting that calaxin tends to adopt the open state in Mg2+-bound form. SAXS data supports that Ca2+-binding causes the structural transition toward the closed state. The changes in the structural transition of the C-terminal domain may be required to bind outer-arm dynein. These results provide a novel mechanism for recognizing a target protein using a calcium sensor protein

A new target region for changing the substrate specificity of amine transaminases

(R)-stereospecific amine transaminases (R-ATAs) are important biocatalysts for the production of (R)-amine compounds in a strict stereospecific manner. An improved R-ATA, ATA-117-Rd11, was successfully engineered for the manufacture of sitagliptin, a widely used therapeutic agent for type-2 diabetes. The effects of the individual mutations, however, have not yet been demonstrated due to the lack of experimentally determined structural information. Here we describe three crystal structures of the first isolated R-ATA, its G136F mutant and engineered ATA-117-Rd11, which indicated that the mutation introduced into the 136th residue altered the conformation of a loop next to the active site, resulting in a substrate-binding site with drastically modified volume, shape, and surface properties, to accommodate the large pro-sitagliptin ketone. Our findings provide a detailed explanation of the previously reported molecular engineering of ATA-117-Rd11 and propose that the loop near the active site is a new target for the rational design to change the substrate specificity of ATAs.UTokyo Research掲載「さまざまな薬の合成に重要な酵素が作用する相手を認識する仕組み」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/how-an-important-enzyme-used-in-drug-production-recognizes-its-substrate.htmlUTokyo Research "How an important enzyme used in drug production recognizes its substrate" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/how-an-important-enzyme-used-in-drug-production-recognizes-its-substrate.htm