High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase.

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Šresolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

Charge-density analysis of an iron-sulfur protein at an ultra-high resolution of 0.48 Å.

The fine structures of proteins, such as the positions of hydrogen atoms, distributions of valence electrons and orientations of bound waters, are critical factors for determining the dynamic and chemical properties of proteins. Such information cannot be obtained by conventional protein X-ray analyses at 3.0-1.5 Å resolution, in which amino acids are fitted into atomically unresolved electron-density maps and refinement calculations are performed under strong restraints. Therefore, we usually supplement the information on hydrogen atoms and valence electrons in proteins with pre-existing common knowledge obtained by chemistry in small molecules. However, even now, computational calculation of such information with quantum chemistry also tends to be difficult, especially for polynuclear metalloproteins. Here we report a charge-density analysis of the high-potential iron-sulfur protein from the thermophilic purple bacterium Thermochromatium tepidum using X-ray data at an ultra-high resolution of 0.48 Å. Residual electron densities in the conventional refinement are assigned as valence electrons in the multipolar refinement. Iron 3d and sulfur 3p electron densities of the Fe4S4 cluster are visualized around the atoms. Such information provides the most detailed view of the valence electrons of the metal complex in the protein. The asymmetry of the iron-sulfur cluster and the protein environment suggests the structural basis of charge storing on electron transfer. Our charge-density analysis reveals many fine features around the metal complex for the first time, and will enable further theoretical and experimental studies of metalloproteins.

Synthesis of crosslinked 2'-OMe RNA duplexes and their application for effective inhibition of miRNA function.

The interstrand crosslinking of nucleic acids is one of the strategies to create the stable complex between an oligonucleotide and RNA by covalent bond formation. We previously reported that fully 2'-O-methylated (2'-OMe) RNAs having the 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. In this study, we established a chemical method to efficiently synthesize the crosslinked 2'-OMe RNA duplexes using AVP and prepared the anti-miRNA oligonucleotides (AMOs) containing the antisense targeting miR-21 and crosslinked duplex at the terminal sequences. These AMOs showed a markedly higher anti miRNA activity than that of the commercially-available miR-21 inhibitor which has locked nucleic acid (LNA) residues.

Structure of the LH1-RC complex from Thermochromatium tepidum at 3.0 Å.

The light-harvesting core antenna (LH1) and the reaction centre (RC) of purple photosynthetic bacteria form a supramolecular complex (LH1-RC) to use sunlight energy in a highly efficient manner. Here we report the first near-atomic structure, to our knowledge, of a LH1-RC complex, namely that of a Ca(2+)-bound complex from Thermochromatium tepidum, which reveals detailed information on the arrangement and interactions of the protein subunits and the cofactors. The RC is surrounded by 16 heterodimers of the LH1 αß-subunit that form a completely closed structure. The Ca(2+) ions are located at the periplasmic side of LH1. Thirty-two bacteriochlorophyll and 16 spirilloxanthin molecules in the LH1 ring form an elliptical assembly. The geometries of the pigment assembly involved in the absorption characteristics of the bacteriochlorophyll in LH1 and excitation energy transfer among the pigments are reported. In addition, possible ubiquinone channels in the closed LH1 complex are proposed based on the atomic structure.

Analysis of time-course drug response in rat cardiomyocytes cultured on a pattern of islands.

We previously reported the kinetics analysis of cardiomyocyte beating using scanning electrochemical microscopy (SECM). In this study, a stage-top incubator and a capillary micropipette (MP) for delivering drugs were assembled with an SECM instrument, and the responses of rat cardiomyocytes were analyzed under a culture environment after drug stimulation. When adenosine triphosphate (ATP) was delivered to synchronously beating cardiomyocytes, the beating acceleration effect of ATP was counteracted by the synchronously beating network in the culture dish. In contrast, cardiomyocytes cultured on a pattern of islands in a culture dish showed fluctuations in the duration of beating upon the addition of ATP. We also examined the effect of the cardiotoxic agent astemizole on cardiomyocytes and successfully detected motion fluctuations. Therefore, drug stimulation via MPs and beating measurement by SECM are effective routes for the evaluation of drug candidates through the analysis of time-course beating motion fluctuations of the cardiomyocytes.

Establishment of estrogen receptor 1 (ESR1)-knockout medaka: ESR1 is dispensable for sexual development and reproduction in medaka, Oryzias latipes.

Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptor (ESR) in all vertebrates. Most vertebrates have two ESR subtypes (ESR1 and ESR2), whereas teleost fish have at least three (Esr1, Esr2a and Esr2b). Intricate functionalization has been suggested among the Esr subtypes, but to date, distinct roles of Esr have been characterized in only a limited number of species. Study of loss-of-function in animal models is a powerful tool for application to understanding vertebrate reproductive biology. In the current study, we established esr1 knockout (KO) medaka using a TALEN approach and examined the effects of Esr1 ablation. Unexpectedly, esr1 KO medaka did not show any significant defects in their gonadal development or in their sexual characteristics. Neither male or female esr1 KO medaka exhibited any significant changes in sexual differentiation or reproductive activity compared with wild type controls. Interestingly, however, estrogen-induced vitellogenin gene expression, an estrogen-responsive biomarker in fish, was limited in the liver of esr1 KO males. Our findings, in contrast to mammals, indicate that Esr1 is dispensable for normal development and reproduction in medaka. We thus provide an evidence for estrogen receptor functionalization between mammals and fish. Our findings will also benefit interpretation of studies into the toxicological effects of estrogenic chemicals in fish.

High-resolution crystal structures of the solubilized domain of porcine cytochrome b5.

Mammalian microsomal cytochrome b5 has multiple electron-transfer partners that function in various electron-transfer reactions. Four crystal structures of the solubilized haem-binding domain of cytochrome b5 from porcine liver were determined at sub-angstrom resolution (0.76-0.95 Å) in two crystal forms for both the oxidized and reduced states. The high-resolution structures clearly displayed the electron density of H atoms in some amino-acid residues. Unrestrained refinement of bond lengths revealed that the protonation states of the haem propionate group may be involved in regulation of the haem redox properties. The haem Fe coordination geometry did not show significant differences between the oxidized and reduced structures. However, structural differences between the oxidized and reduced states were observed in the hydrogen-bond network around the axial ligand His68. The hydrogen-bond network could be involved in regulating the redox states of the haem group.

Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy.

The contractile behavior of cardiomyocytes can be monitored by measuring their action potentials, and the analysis is essential for screening the safety of potential drugs. However, immobilizing cardiac cells on a specific electrode is considerably complicated. In this study, we demonstrate that scanning electrochemical microscopy (SECM) can be used to analyze rapid topographic changes in beating cardiomyocytes in a standard culture dish. Various cardiomyocyte contraction parameters and oxygen consumption based on cell respiration could be determined from SECM data. We also confirmed that cellular changes induced by adding the cardiotonic agent digoxin were conveniently monitored by this SECM system. These results show that SECM can be a potentially powerful tool for use in drug development for cardiovascular diseases.

Description of peptide bond planarity from high-resolution neutron crystallography.

Neutron crystallography is a highly effective method for visualizing hydrogen atoms in proteins. In our recent study, we successfully determined the high-resolution (1.2 Å) neutron structure of high-potential iron-sulfur protein, refining the coordinates of some amide protons without any geometric restraints. Interestingly, we observed that amide protons are deviated from the peptide plane due to electrostatic interactions. Moreover, the difference in the position of the amide proton of Cys75 between reduced and oxidized states is possibly attributed to the electron storage capacity of the iron-sulfur cluster. Additionally, we have discussed about the rigidity of the iron-sulfur cluster based on the results of the hydrogen-deuterium exchange. Our research underscores the significance of neutron crystallography in protein structure elucidation, enriching our understanding of protein functions at an atomic resolution.

Activation of oxidoreductases by the formation of enzyme assembly.

Biological properties of protein molecules depend on their interaction with other molecules, and enzymes are no exception. Enzyme activities are controlled by their interaction with other molecules in living cells. Enzyme activation and their catalytic properties in the presence of different types of polymers have been studied in vitro, although these studies are restricted to only a few enzymes. In this study, we show that addition of poly-l-lysine (PLL) can increase the enzymatic activity of multiple oxidoreductases through formation of enzyme assemblies. Oxidoreductases with an overall negative charge, such as l-lactate oxidase, d-lactate dehydrogenase, pyruvate oxidase, and acetaldehyde dehydrogenase, each formed assemblies with the positively charged PLL via electrostatic interactions. The enzyme activities of these oxidoreductases in the enzyme assemblies were several-folds higher than those of the enzyme in their natural dispersed state. In the presence of PLL, the turnover number (kcat) improved for all enzymes, whereas the decrease in Michaelis constant (KM) was enzyme dependent. This type of enzyme function regulation through the formation of assemblies via simple addition of polymers has potential for diverse applications, including various industrial and research purposes.

New insights into the oxidation process from neutron and X-ray crystal structures of an O2-sensitive [NiFe]-hydrogenase.

[NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an O2-sensitive enzyme that is inactivated in the presence of O2 but the oxidized enzyme can recover its catalytic activity by reacting with H2 under anaerobic conditions. Here, we report the first neutron structure of [NiFe]-hydrogenase in its oxidized state, determined at a resolution of 2.20 Å. This resolution allowed us to reinvestigate the structure of the oxidized active site and to observe the positions of protons in several short hydrogen bonds. X-ray anomalous scattering data revealed that a part of the Ni ion is dissociated from the active site Ni-Fe complex and forms a new square-planar Ni complex, accompanied by rearrangement of the coordinated thiolate ligands. One of the thiolate Sγ atoms is oxidized to a sulfenate anion but remains attached to the Ni ion, which was evaluated by quantum chemical calculations. These results suggest that the square-planar complex can be generated by the attack of reactive oxygen species derived from O2, as distinct from one-electron oxidation leading to a conventional oxidized form of the Ni-Fe complex. Another major finding of this neutron structure analysis is that the Cys17S thiolate Sγ atom coordinating to the proximal Fe-S cluster forms an unusual hydrogen bond with the main-chain amide N atom of Gly19S with a distance of 3.25 Å, where the amide proton appears to be delocalized between the donor and acceptor atoms. This observation provides insight into the contribution of the coordinated thiolate ligands to the redox reaction of the Fe-S cluster.

Structure analysis and comparative characterization of the cytochrome c' and flavocytochrome c from thermophilic purple photosynthetic bacterium Thermochromatium tepidum.

The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22 °C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins.

Promotion of cytoplasmic localization of oligonucleotides by connecting cross-linked duplexes.

We previously reported that antisense oligonucleotides (ASOs) flanked by duplexes can suppress microRNA (miRNA) function with high efficiency for a long duration. In this study, we examined the effect of the double-stranded structure on the subcellular localization of ASOs. Double strands were cross-linked to prevent dissociation into single strands, and this cross-linked duplex (CD) was connected at the 5' or 3' termini of an antisense-targeting miRNA-21 (AS). The subcellular distribution of fluorescently labelled ASOs was analyzed following transfection into cells. While single-stranded AS molecules promptly moved to the nucleus, AS with the CD at the 5'-end (5'CD-AS) interestingly showed significantly higher cytoplasmic localization. The 3'-CD-modified AS (3'CD-AS) was degraded from the 5'-end of the AS, but the degradation was prevented by 5'-end chemical modifications, thereby allowing the imaging of the cytoplasmic localization. The CD modification significantly promoted the cytoplasmic localization of ASOs and enabled the effective knockdown of miRNA existing in cytoplasm. These results reveal that the duplex structure has promising potential to control the subcellular distribution of ASOs.

Interspecific differences in the responses of root phosphatase activities and morphology to nitrogen and phosphorus fertilization in Bornean tropical rain forests.

Soil organic phosphorus (P) compounds can be the main P source for plants in P-limited tropical rainforests. Phosphorus occurs in diverse chemical forms, including monoester P, diester P, and phytate, which require enzymatic hydrolysis by phosphatase into inorganic P before assimilation by plants. The interactions between plant interspecific differences in organic P acquisition strategies via phosphatase activities with root morphological traits would lead to P resource partitioning, but they have not been rigorously evaluated. We measured the activities of three classes of phosphatases (phosphomonoesterase, PME; phosphodiesterase, PDE; and phytase, PhT), specific root length (SRL), root diameter, and root tissue density in mature tree species with different mycorrhizal associations (ectomycorrhizal [ECM] or arbuscular mycorrhizal [AM]) and different successional status (climax or pioneer species) in Sabah, Malaysia. We studied nitrogen (N)- and P-fertilized plots to evaluate the acquisition strategies for organic P under P-limited conditions 7 years after fertilization was initiated. P fertilization reduced the PME activity in all studied species and reduced PhT and PDE activities more in climax species than in the two pioneer species, irrespective of the mycorrhizal type. PDE activity increased in some climax species after N fertilization, suggesting that these species allocate excess N to the synthesis of PDE. Moreover, PME and PhT activities, but not PDE activity, correlated positively with SRL. We suggest that climax species tend to be more strongly dependent on recalcitrant organic P (i.e., phytate and/or diester P) than pioneer species, regardless of the mycorrhizal type. We also suggest that trees in which root PME or PhT activity is enhanced can increase their SRL to acquire P efficiently. Resource partitioning of soil organic P would occur among species through differences in their phosphatase activities, which plays potentially ecologically important role in reducing the competition among coexisting tree species in lowland tropical rainforests.

Revisiting the concept of peptide bond planarity in an iron-sulfur protein by neutron structure analysis.

The planarity of the peptide bond is important for the stability and structure formation of proteins. However, substantial distortion of peptide bonds has been reported in several high-resolution structures and computational analyses. To investigate the peptide bond planarity, including hydrogen atoms, we report a 1.2-Šresolution neutron structure of the oxidized form of high-potential iron-sulfur protein. This high-resolution neutron structure shows that the nucleus positions of the amide protons deviate from the peptide plane and shift toward the acceptors. The planarity of the H─N─C═O plane depends strongly on the pyramidalization of the nitrogen atom. Moreover, the orientation of the amide proton of Cys75 is different in the reduced and oxidized states, possibly because of the electron storage capacity of the iron-sulfur cluster.

Synthesis of Crosslinked 2'-OMe RNA Duplexes Using 2-Amino-6-Vinylpurine and Their Application for Effective Inhibition of miRNA Function.

Crosslinking reactions to nucleic acids are an effective way to prepare stable complexes formed by covalent bonding. We demonstrated that fully 2'-O-methylated (2'-OMe) RNAs having a 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. Recently, we reported the preparation of crosslinked 2'-OMe RNA duplexes using AVP and the anti-miRNA oligonucleotides (AMOs) containing crosslinked duplexes at the terminal positions. These AMOs exhibited efficient microRNA (miRNA) inhibition at very low concentrations. In this article, we describe the chemical synthesis of 2'-OMe oligonucleotides containing AVP and preparation of the AMOs bearing crosslinked 2'-OMe RNA duplexes using AVP. In addition, we describe in detail the miRNA inhibition assay using these AMOs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phosphoramidite of 2-amino-6-vinylguanosine derivative Basic Protocol 2: Synthesis of AVP-2'-OMe RNA Basic Protocol 3: Evaluation of the crosslink reactivity of CFO containing AVP to the 2'-OMe RNA and preparation of AMOs containing crosslinked duplex Basic Protocol 4: miRNA inhibition assays.

Dimer structure and conformational variability in the N-terminal region of an archaeal small heat shock protein, StHsp14.0.

Small heat shock proteins (sHsps), which are categorized into a class of molecular chaperones, bind and stabilize denatured proteins to prevent aggregation. The sHsps undergo transition between different oligomeric states to control their hydrophobicity. So far, only the structures of sHsps in large oligomeric states have been reported. Here we report the structure of StHsp14.0 from Sulfolobus tokodaii in the dimeric state, which is formed by means of a mutation at the C-terminal IXI/V motif. The dimer is the sole building block in two crystal forms, and the dimeric mode is the same as that in the large oligomers. The N-terminal helix has variety in its conformation. Furthermore, spectroscopic and biochemical experiments were performed to investigate the conformational variability at the N-terminus. The structural, dynamical and oligomeric properties suggest that chaperone activity of StHsp14.0 is mediated by partially dissolved oligomers.

Stable duplex-linked antisense targeting miR-148a inhibits breast cancer cell proliferation.

MicroRNAs (miRNAs) regulate cancer cell proliferation by binding directly to the untranslated regions of messenger RNA (mRNA). MicroRNA-148a (miR-148a) is expressed at low levels in breast cancer (BC). However, little attention has been paid to the sequestration of miR-148a. Here, we performed a knockdown of miR-148a using anti-miRNA oligonucleotides (AMOs) and investigated the effect on BC cell proliferation. BC cell proliferation was significantly suppressed by AMO flanked by interstrand cross-linked duplexes (CL-AMO), whereas single-stranded and commercially available AMOs had no effect. The suppression was caused by sequestering specifically miR-148a. Indeed, miR-148b, another member of the miR-148 family, was not affected. Importantly, the downregulation of miR-148a induced a greater and longer-lasting inhibition of BC cell proliferation than the targeting of oncogenic microRNA-21 (miR-21) did. We identified thioredoxin-interacting protein (TXNIP), a tumor suppressor gene, as a target of miR-148a and showed that CL-AMO provoked an increase in TXNIP mRNA expression. This study provide evidence that lowly expressed miRNAs such as miR-148a have an oncogenic function and might be a promising target for cancer treatment.

Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans.

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af-Tth) was determined by X-ray crystallography to a resolution of 1.95 Å. Af-Tth is a homodimer, and its monomer structure exhibits an eight-bladed ß-propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the ß-propeller region. We observed unexplained electron densities in this cavity of the substrate-soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site-specific Af-Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af-Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step.

Detailed assessment of X-ray induced structural perturbation in a crystalline state protein.

The positions of hydrogen atoms significantly define protein functions. However, such information from protein crystals is easily disturbed by X-rays. The damage can not be prevented completely even in the data collection at cryogenic temperatures. Therefore, the influence of X-rays should be precisely estimated in order to derive meaningful information from the crystallographic results. Diffraction data from a single crystal of the high-potential iron-sulfur protein (HiPIP) from Thermochromatium tepidum were collected at an undulator beamline of a third generation synchrotron facility, and were merged into three data sets according to X-ray dose. A series of structures analyzed at 0.70A shows detailed views of the X-ray induced perturbation, such as the positional changes of hydrogen atoms of a water molecule. Based on the results, we successfully collected a low perturbation data set using attenuated X-rays. There was no influence on the crystallographic statistics, such as the relative B factors, during the course of data collection. The electron densities for hydrogen atoms were more clear despite the slightly lower resolution.