Structures and mechanisms of actin ATP hydrolysis.

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Three-Component Synthesis of Quinoline-4-carboxylic Acids Based on Doebner Hydrogen-Transfer Reaction.

The Doebner hydrogen-transfer reaction has been developed for the synthesis of substituted quinolines from anilines possessing electron-withdrawing groups, which are known to give products in low yields when used in the conventional Doebner reaction. This reaction can be applied to not only anilines having electron-withdrawing groups but also those having electron-donating groups and can be used in the large-scale synthesis of bioactive molecules.

2,3-Butandione 2-monoxime inhibits skeletal myosin II by accelerating ATP cleavage.

2,3-Butandione 2-monoxime (BDM) is a widely used myosin inhibitor with an unclear mode of action. In this report, we investigated the mechanism of BDM oxime group nucleophilic reactivity on the phosphoester bond of ATP. BDM increased the ATPase activity of skeletal myosin subfragment 1 (S1) under conditions in which ATP cleavage is the rate-limiting step (K+, EDTA-ATPase activity of native S1 and Mg2+-ATPase activity of trinitrophenylated S1 and partially unfolded S1). Furthermore, the effect of BDM on the S1-bound adenosine 5'-(ß,γ-imido) triphosphate (AMPPNP) 31P NMR spectrum suggests that BDM changes the microenvironment around the phosphorus atoms of myosin-bound nucleotide. A computational search for the BDM-binding site in the adenosine 5'-[γ-thio] triphosphate (myosin-ATPγS) complex predicted that BDM is located adjacent to the nucleotide on myosin. Therefore, we propose that the BDM oxime group catalytically assists in ATP cleavage, thereby enhancing the ATPase activity of myosin in a manner analogous to pralidoxime-mediated reactivation of organophosphate-inactivated acetylcholinesterase. This is the first study suggesting that oxime provides catalytic assistance for ATP cleavage by an ATP-hydrolyzing enzyme.

Phase transition kinetics of LiNi0.5Mn1.5O4 analyzed by temperature-controlled operando X-ray absorption spectroscopy.

LiNi0.5Mn1.5O4 (LNMO) is a promising positive electrode material for lithium ion batteries because it shows a high potential of 4.7 V vs. Li/Li(+). Its charge-discharge reaction includes two consecutive phase transitions between LiNi0.5Mn1.5O4 (Li1) ↔ Li0.5Ni0.5Mn1.5O4 (Li0.5) and Li0.5 ↔ Ni0.5Mn1.5O4 (Li0) and the complex transition kinetics that governs the rate capability of LNMO can hardly be analyzed by simple electrochemical techniques. Herein, we apply temperature-controlled operando X-ray absorption spectroscopy to directly capture the reacting phases from -20 °C to 40 °C under potential step (chronoamperometric) conditions and evaluate the phase transition kinetics using the apparent first-order rate constants at various temperatures. The constant for the Li1 ↔ Li0.5 transition (process 1) is larger than that for the Li0.5 ↔ Li0 transition (process 2) at all the measured temperatures, and the corresponding activation energies are 29 and 46 kJ mol(-1) for processes 1 and 2, respectively. The results obtained are discussed to elucidate the limiting factor in this system as well as in other electrode systems.

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples.

We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time.

Development of a heme sensor using fluorescently labeled heme oxygenase-1.

Free heme, the protein-unbound form of heme, has both toxic and regulatory effects on cells. To detect free heme at low concentrations, we developed a heme sensor using fluorescently labeled heme oxygenase-1 (HO-1), an enzyme that catalyzes oxidative heme degradation and has a high affinity for heme. The response of the heme sensor is based on the fluorescence quenching that occurs when heme binds to the enzyme. Each of the three fluorescently labeled HO-1s exhibits a 1:1 binding stoichiometry and an absorption spectrum similar to that of the heme complex of the wild-type HO-1. Titration of the labeled proteins with hemin resulted in fluorescence quenching in a hemin concentration-dependent manner, presumably due to an energy transfer from the fluorophore to the heme bound to HO-1. The sensor showed a potent affinity for heme with a dissociation constant in the low nanomolar range and a high selectivity for heme. Based on the linear response of the sensor to heme, we performed a fluorometric microplate assay. The sensor was able to selectively detect free heme but did not respond to heme bound to native hemoglobin. This assay will be a useful tool for determination of free heme in biological samples containing protein-bound heme.

Identification of compounds with potential antibacterial activity against Mycobacterium through structure-based drug screening.

To identify novel antibiotics against Mycobacterium tuberculosis, we performed a hierarchical structure-based drug screening (SBDS) targeting the enoyl-acyl carrier protein reductase (InhA) with a compound library of 154,118 chemicals. We then evaluated whether the candidate hit compounds exhibited inhibitory effects on the growth of two model mycobacterial strains: Mycobacterium smegmatis and Mycobacterium vanbaalenii. Two compounds (KE3 and KE4) showed potent inhibitory effects against both model mycobacterial strains. In addition, we rescreened KE4 analogs, which were identified from a compound library of 461,383 chemicals through fingerprint analysis and genetic algorithm-based docking simulations. All of the KE4 analogs (KES1-KES5) exhibited inhibitory effects on the growth of M. smegmatis and/or M. vanbaalenii. Based on the predicted binding modes, we probed the structure-activity relationships of KE4 and its analogs and found a correlative relationship between the IC50 values and the interaction residues/LogP values. The most potent inhibitor, compound KES4, strongly and stably inhibited the long-term growth of the model bacteria and showed higher inhibitory effects (IC50 = 4.8 µM) than isoniazid (IC50 = 5.4 µM), which is a first-line drug for tuberculosis therapy. Moreover, compound KES4 did not exhibit any toxic effects that impede cell growth in several mammalian cell lines and enterobacteria. The structural and experimental information of these novel chemical compounds will likely be useful for the development of new anti-TB drugs. Furthermore, the methodology that was used for the identification of the effective chemical compound is also likely to be effective in the SBDS of other candidate medicinal drugs.

A luminescent affinity tag for proteins based on the terbium(III)-binding peptide.

Genetically encoded tags attached to proteins of interest are widely exploited for proteome analysis. Here, we present Tb(3+)-binding peptides (TBPs) which can be used for both luminescent measurements and affinity purification of proteins. TBPs consist of acidic amino acid residues and tryptophan residues which serve as Tb(3+)-binding sites and sensitizers for Tb(3+) luminescence, respectively. The Tb(3+) complexes of TBPs fused to a target protein exhibited luminescence characteristic of Tb(3+) by excitation of the tryptophan residue, and fusion proteins fused to one of the TPBs were successfully isolated from Escherichia coli cell lysate by affinity chromatography with a Tb(3+)-immobilized solid support.

Discovery of novel Li SSE and anode coatings using interpretable machine learning and high-throughput multi-property screening.

All-solid-state batteries with Li metal anode can address the safety issues surrounding traditional Li-ion batteries as well as the demand for higher energy densities. However, the development of solid electrolytes and protective anode coatings possessing high ionic conductivity and good stability with Li metal has proven to be a challenge. Here, we present our informatics approach to explore the Li compound space for promising electrolytes and anode coatings using high-throughput multi-property screening and interpretable machine learning. To do this, we generate a database of battery-related materials properties by computing [Formula: see text] migration barriers and stability windows for over 15,000 Li-containing compounds from Materials Project. We screen through the database for candidates with good thermodynamic and electrochemical stabilities, and low [Formula: see text] migration barriers, identifying promising new candidates such as [Formula: see text]N, [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text], among others. We train machine learning models, using ensemble methods, to predict migration barriers and oxidation and reduction potentials of these compounds by engineering input features that ensure accuracy and interpretability. Using only a small number of features, our gradient boosting regression models achieve [Formula: see text] values of 0.95 and 0.92 on the oxidation and reduction potential prediction tasks, respectively, and 0.86 on the migration barrier prediction task. Finally, we use Shapley additive explanations and permutation feature importance analyses to interpret our machine learning predictions and identify materials properties with the largest impact on predictions in our models. We show that our approach has the potential to enable rapid discovery and design of novel solid electrolytes and anode coatings.

Phosphorylation of clustered serine residues in the N-terminus of BPS domain negatively regulates formation of the complex between human Grb14 and insulin receptor.

Growth factor receptor-bound protein 14 (Grb14) is a negative regulator of insulin receptor (IR) and is involved in a negative feedback mechanism of insulin signaling. Grb14 associates with IR and inhibits its tyrosine kinase activity through the between pleckstrin homology and Src homology-2 (BPS) domain. We previously reported that the pharmacological inhibition and knockdown of glycogen synthase kinase-3 (GSK-3) facilitates the insulin-induced complex formation of human Grb14 (hGrb14) and IR, suggesting that GSK-3 suppresses hGrb14 recruitment to IR. This study further investigated a functional phosphorylation of the serine residues in hGrb14 BPS domain, identified as putative GSK-3 targets to verify an effect of GSK-3 on the hGrb14-IR complex formation. In vitro kinase assay using the motif-derived peptides showed that the serine residues located in N-terminal (Ser358, Ser362 and Ser366) and C-terminal (Ser419 and Ser423) regions of the BPS domain were phosphorylated by GSK-3. Co-immunoprecipitation and yeast two-hybrid (Y2H) experiments suggested that the negative charges genetically introduced on the Ser358, Ser362 and Ser366 suppressed the association of hGrb14 to IR. Surface plasmon resonance experiment gave Kd values of 8 nM for recombinant hGrb14 with respect to the interaction with IR ß-subunit, and this affinity was lost after the replacements of the Ser358, Ser362 and Ser366 with glutamic acid residues. Y2H experiment with the BPS domain alone; however, did not show any difference owing to the same mutations. It is therefore evident that the N-terminus of the BPS domain plays an important role in the regulation of hGrb14-IR complex formation through phosphorylation, in addition to other domains.

Identification of a novel class of small compounds with anti-tuberculosis activity by in silico structure-based drug screening.

The enzymes responsible for biotin biosynthesis in mycobacteria have been considered as potential drug targets owing to the important role in infection and cell survival that the biotin synthetic pathway plays in Mycobacterium tuberculosis. Among the enzymes that comprise mycobacterium biotin biosynthesis systems, 7,8-diaminopelargonic acid synthase (DAPAS) plays an essential role during the stationary phase in bacterial growth. In this study, compounds that inhibit mycobacterial DAPAS were screened in the virtual chemical library using an in silico structure-based drug screening (SBDS) technique, and the antimycobacterial activity of the selected compounds was validated experimentally. The DOCK-GOLD programs utilized by in silico SBDS facilitated the identification of a compound, referred to as KMD6, with potent inhibitory effects on the growth of model mycobacteria (M. smegmatis). The subsequent compound search, which was based on the structural features of KMD6, resulted in identification of three additional active compounds, designated as KMDs3, KMDs9 and KMDs10. The inhibitory effect of these compounds was comparable to that of isoniazid, which is a first-line antituberculosis drug. The high antimycobacterial activity of KMD6, KMDs9 and KMDs10 was maintained on the experiment with M. tuberculosis. Of the active compounds identified, KMDs9 would be a promising pharmacophore, owing to its long-term antimycobacterial effect and lack of cytotoxicity.

In silico structure-based drug screening of novel antimycobacterial pharmacophores by DOCK-GOLD tandem screening.

BACKGROUND: Enzymes responsible for cell wall development in Mycobacterium tuberculosis are considered as potential targets of anti-tuberculosis (TB) agents. Mycobacterial cyclopropane mycolic acid synthase 1 (CmaA1) is essential for mycobacterial survival because of its critical role in synthesizing mycolic acids. MATERIALS AND METHODS: We screened compounds that were capable of interacting with the mycobacterial CmaA1 active site using a virtual compound library with an in silico structure-based drug screening (SBDS). Following the selection of such compounds, their antimycobacterial activity was examined. RESULTS: With the in silico SBDS, for which we also used DOCK-GOLD programs and screening methods that utilized the structural similarity between the selected active compounds, we identified two compounds with potent inhibitory effects on mycobacterial growth. The antimycobacterial effect of the compounds was comparable to that of isoniazid, which is used as a first-line anti-TB drug. CONCLUSION: The compounds identified through SBDS were expected to be a novel class of anti-TB pharmacophores.

Aggregation of partially unfolded Myosin subfragment-1 into spherical oligomers with amyloid-like dye-binding properties.

Proteolytic myosin subfragment 1 (S1) is known to be partially unfolded in its 50-kDa subdomain by mild heat treatment at 35 degrees C [Burke et al. (1987) Biochemistry 26, 1492-1496]. Here, we report that this partial unfolding is accompanied by aggregation of S1 protein. Characteristics of the aggregate thus formed were: (i) formation of transparent sediment under centrifugation at 183,000 x g; (ii) amyloid-like, dye-binding properties such as Congo red-binding and Thioflavin T fluorescence enhancement; (iii) a uniformly sized spherical appearance in electron micrographs; and (iv) sensitivity to tryptic digestion. Gel filtration analysis of the aggregation process indicates that the spheroid was formed through an intermediate oligomeric stage. The aggregate inhibited spontaneous aggregation of an isolated 50 kDa fragment into a large amorphous mass. The remaining native regions in the partially unfolded S1 were probably responsible for this effect. These results show that, unlike the 50-kDa fragment, the partially unfolded S1 molecules do not form amorphous aggregates but assemble into spherical particles. The native regions in partially unfolded S1 may be a determinant of aggregate morphology.

Structural Understanding of Superior Battery Properties of Partially Ni-Doped Li2MnO3 as Cathode Material.

We examined the crystal structures of Li2(NixMn1-x)O3(-δ) (x = 0, 1/10, 1/6, and 1/4) to elucidate the relationship between the structure and electrochemical performance of the compounds using neutron and synchrotron X-ray powder diffraction analyses in combination. Our examination revealed that these crystals contain a large number of stacking faults and exhibit significant cation mixing in the transition-metal layers; the cation mixing becomes significant with an increase in the Ni concentration. Charge-discharge measurements showed that the replacement of Mn with Ni lowers the potential of the charge plateau and leads to higher charge-discharge capacities. From a topological point of view with regard to the atomic arrangement in the crystals, it is concluded that substituting Mn in Li2MnO3 with Ni promotes the formation of smooth Li percolation paths, thus increasing the number of active Li ions and improving the charge-discharge capacity.

Discovery of InhA inhibitors with anti-mycobacterial activity through a matched molecular pair approach.

The Mycobacterium tuberculosis (M. tuberculosis) enoyl-acyl carrier protein reductase (mtInhA) is an attractive enzyme and a thoroughly studied target for tuberculosis therapy. In this study, to identify novel structure-activity relationships (SARs) of mtInhA inhibitors, a series of diphenyl ether derivatives were designed based on the matched molecular pair (MMP) method, and the binding energies of these compounds were subsequently estimated by in silico structure-based drug screening (SBDS) to provide more useful data. Consequently, the 10 unique candidate compounds (KEM1-KEM10) were identified and assessed for the inhibition of mtInhA enzymatic activity, in vitro antibiotic effects against model mycobacteria and toxicity level on both intestinal bacteria and mammalian cells. Among the compounds tested, phenyl group (KEM4) and 2-fluorobenzyl group (KEM7) substitutions produced preferable inhibitory effects on mtInhA enzymatic activity relative to those provided by a furyl group (KES4: base compound) at the terminal of the compound, and KEM7 inhibited the growth of the mycobacteria strain with a lower IC50 value. Moreover, most of the candidate compounds exhibited neither inhibition of the growth of enterobacteria nor toxic effects on mammalian cells, though KEM10 exhibited toxicity against cultured MDCK cells. The structural and experimental information concerning these mtInhA inhibitors identified through MMP-based in silico screening will likely contribute to the lead optimisation of novel antibiotics for M. tuberculosis.

Oxidative rearrangement of spiro cyclobutane cyclic aminals: efficient construction of bicyclic amidines.

A new rearrangement reaction of spirocyclic cyclobutane N-halo aminals is described. This process, promoted by treatment of the aminals with N-halosuccinimides (NXS, X = Br or Cl), efficiently produces bicyclic amidines by a pathway involving initial N-halogenation of one of the aminal nitrogens followed by cyclobutane ring expansion through 1,2-C-to-N migration with simultaneous N-X bond cleavage.

Kinetics of dextran-independent α-(1→3)-glucan synthesis by Streptococcus sobrinus glucosyltransferase I.

Glucosyltransferase (GTF)-I from cariogenic Streptococcus sobrinus elongates the α-(1→3)-linked glucose polymer branches on the primer dextran bound to the C-terminal glucan-binding domain. We investigated the GTF-I-catalyzed glucan synthesis reaction in the absence of the primer dextran. The time course of saccharide production during dextran-independent glucan synthesis from sucrose was analyzed. Fructose and glucose were first produced by the sucrose hydrolysis. Leucrose was subsequently produced, followed by insoluble glucan [α-(1→3)-linked glucose polymers] after a lag phase. High levels of intermediate nigerooligosaccharide series accumulation were characteristically not observed during the lag phase. The results from the enzymatic activity of the acceptor reaction for the nigerooligosaccharide with a degree of polymerization of 2-6 and methyl α-D-glucopyranoside as a glucose analog indicate that the activity increased with an increase in the degree of polymerization. The production of insoluble glucan was numerically simulated using the fourth-order Runge-Kutta method with the kinetic parameters estimated from the enzyme assay. The simulated time course provided a profile similar to that of experimental data. These results define the relationship between the kinetic properties of GTF-I and the time course of saccharide production. These results are discussed with respect to a mechanism that underlies efficient glucan synthesis.

Facile preparation of oxazole-4-carboxylates and 4-ketones from aldehydes using 3-oxazoline-4-carboxylates as intermediates.

A novel 2-step synthesis of oxazole-4-carboxylates from aldehydes was developed, which is characterized by the utilization of 3-oxazoline-4-carboxylates as synthetic intermediates. The facile preparation of 4-keto-oxazole derivatives from 3-oxazoline-4-carboxylates based on their interesting reactivity toward Grignard reagents is also described.

Organic synthesis using a hypervalent iodine reagent: unexpected and novel domino reaction leading to spiro cyclohexadienone lactones.

The reaction of 1-(p-hydroxyaryl)cyclobutanols and phenyl iodide(III) diacetate in hexafluoroisopropanol and water produced spiro cyclohexadienone lactones via a domino reaction.