Direct inhibition of human APOBEC3 deaminases by HIV-1 Vif independent of the proteolysis pathway.

HIV-1 Vif is known to counteract the antiviral activity of human apolipoprotein B mRNA-editing catalytic polypeptide-like (A3), a cytidine deaminase, in various ways. However, the precise mechanism behind this interaction has remained elusive. Within infected cells, Vif forms a complex called VßBCC, comprising CBFß and the components of E3 ubiquitin ligase, Elongin B, Elongin C, and Cullin5. Together with the ubiquitin-conjugating enzyme, VßBCC induces ubiquitination-mediated proteasomal degradation of A3. However, Vif exhibits additional counteractive effects. In this study, we elucidate that VßBCC inhibits deamination by A3G, A3F, and A3B independently of proteasomal degradation. Surprisingly, we discovered that this inhibition for A3G is directly attributed to the interaction between VßBCC and the C-terminal domain of A3G. Previously, it was believed that Vif did not interact with the C-terminal domain. Our findings suggest that inhibiting the interaction between VßBCC and the C-terminal domain, as well as the N-terminal domain known to be targeted for ubiquitination, of A3G may be needed to prevent counteraction by Vif.

NMR characterization of the structure of the intrinsically disordered region of human origin recognition complex subunit 1, hORC1, and of its interaction with G-quadruplex DNAs.

Human origin recognition complex (hORC) binds to the DNA replication origin and then initiates DNA replication. However, hORC does not exhibit DNA sequence-specificity and how hORC recognizes the replication origin on genomic DNA remains elusive. Previously, we found that hORC recognizes G-quadruplex structures potentially formed near the replication origin. Then, we showed that hORC subunit 1 (hORC1) preferentially binds to G-quadruplex DNAs using a hORC1 construct comprising residues 413 to 511 (hORC1413-511). Here, we investigate the structural characteristics of hORC1413-511 in its free and complex forms with G-quadruplex DNAs. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopic studies indicated that hORC1413-511 is disordered except for a short α-helical region in both the free and complex forms. NMR chemical shift perturbation (CSP) analysis suggested that basic residues, arginines and lysines, and polar residues, serines and threonines, are involved in the G-quadruplex DNA binding. Then, this was confirmed by mutation analysis. Interestingly, CSP analysis indicated that hORC1413-511 binds to both parallel- and (3 + 1)-type G-quadruplex DNAs using the same residues, and thereby in the same manner. Our study suggests that hORC1 uses its intrinsically disordered G-quadruplex binding region to recognize parallel-type and (3 + 1)-type G-quadruplex structures at replication origin.

Enzymatic degradation of glucosaminoglucan and cellulase resistance of cellulose nanofiber coated with glucosaminoglucan.

AIMS: Enzymatic degradation of ß-1,4-linked glucose and glucosamine (glucosaminoglucan, GG), which is prepared from Thiothrix nivea and can act as a cellulose-aminating agent with a strong affinity to cellulose, was attempted. METHODS AND RESULTS: A chitosanase-secreting fungal strain was isolated as a GG-degrading microbe. GG was found to be degraded by not only chitosanases but also cellulases. Based on nuclear magnetic resonance spectroscopy, both enzymes were found to produce GlcN-Glc from GG. The cellulases also produced GlcN-Glc-GlcN-Glc as an additional final digest. Furthermore, aminated (GG-coated) cellulose nanofibers exhibited cellulase resistance. The flexibility of GG adsorbed onto a cellulose crystal was almost identical to that of cellulose, as estimated via the molecular dynamics calculations. CONCLUSIONS: The chitosanase and cellulase hydrolyzed the ß-1,4-linkage from Glc to GlcN and were expected to recognize the tetramer and hexamer units of GG depending on their final products. The cellulose nanofibers acquired cellulase resistance via amination with GG, probably because of the lower activity of cellulase to GG than cellulose.

A heterodimeric hyaluronate lyase secreted by the activated sludge bacterium Haliscomenobacter hydrossis.

Haliscomenobacter hydrossis is a filamentous bacterium common in activated sludge. The bacterium was found to utilize hyaluronic acid, and hyaluronate lyase activity was detected in its culture. However, no hyaluronate lyase gene was found in the genome, suggesting the bacterium secretes a novel hyaluronate lyase. The purified enzyme exhibited two bands on SDS-PAGE and a single peak on gel filtration chromatography, suggesting a heterodimeric composition. N-terminal amino acid sequence and mass spectrometric analyses suggested that the subunits are molybdopterin-binding and [2Fe-2S]-binding subunits of a xanthine oxidase family protein. The presence of the cofactors was confirmed using spectrometric analysis. Oxidase activity was not detected, revealing that the enzyme is not an oxidase but a hyaluronate lyase. Nuclear magnetic resonance analysis of the enzymatic digest revealed that the enzyme breaks hyaluronic acid to 3-(4-deoxy-ß-d-gluc-4-enuronosyl)-N-acetyl-d-glucosamine. As hyaluronate lyases (EC 4.2.2.1) are monomeric or trimeric, the enzyme is the first heterodimeric hyaluronate lyase.

Complex Formation of an RNA Aptamer with a Part of HIV-1 Tat through Induction of Base Triples in Living Human Cells Proven by In-Cell NMR.

An RNA aptamer that strongly binds to a target molecule has the potential to be a nucleic acid drug inside living human cells. To investigate and improve this potential, it is critical to elucidate the structure and interaction of RNA aptamers inside living cells. We examined an RNA aptamer for HIV-1 Tat (TA), which had been found to trap Tat and repress its function in living human cells. We first used in vitro NMR to examine the interaction between TA and a part of Tat containing the binding site for trans-activation response element (TAR). It was revealed that two U-A∗U base triples are formed in TA upon binding of Tat. This was assumed to be critical for strong binding. Then, TA in complex with a part of Tat was incorporated into living human cells. The presence of two U-A∗U base triples was also revealed for the complex in living human cells by in-cell NMR. Thus, the activity of TA in living human cells was rationally elucidated by in-cell NMR.

Biophysical Study of the Structure, Dynamics, and Function of Nucleic Acids.

Nucleic acids have essential roles in all biological processes related to genetic information, such as replication, transcription, translation, repair, and recombination [...].

Proton conduction in hydronium solvate ionic liquids affected by ligand shape.

We investigated the ligand dependence of the proton conduction of hydronium solvate ionic liquids (ILs), consisting of a hydronium ion (H3O+), polyether ligands, and a bis[(trifluoromethyl)sulfonyl]amide anion (Tf2N-; Tf = CF3SO2). The ligands were changed from previously reported 18-crown-6 (18C6) to other cyclic or acyclic polyethers, namely, dicyclohexano-18-crown-6 (Dh18C6), benzo-18-crown-6 (B18C6) and pentaethylene glycol dimethyl ether (G5). Pulsed-field gradient spin echo nuclear magnetic resonance results revealed that the protons of H3O+ move faster than those of cyclic 18C6-based ligands but as fast as those of acyclic G5 ligands. Based on these results and density functional theory calculations, we propose that the coordination of a cyclic ether ligand to the H3O+ ion is essential for fast proton conduction in hydronium solvate ILs. Our results attract special interest for many electro- and bio-chemical applications such as electrolyte systems for fuel cells and artificial ion channels for biological cells.

Investigation of the Interaction of Human Origin Recognition Complex Subunit 1 with G-Quadruplex DNAs of Human c-myc Promoter and Telomere Regions.

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413-511 of human ORC subunit 1, hORC1413-511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413-511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413-511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413-511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413-511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure.

[Successful Conservative Management of Bronchopleural Fistula after Intraoperative Bronchial Injury].

A 64-year-old woman diagnosed as primary lung cancer was admitted for surgery. Right lower lobectomy and ND2a-1 nodal dissection was performed under video-assisted thoracic surgery( VATS). The membranous portion of intermediate bronchus was injured about length of 5 mm while dissecting subcarinal lymph nodes. The fistula was closed by knotted suture using 4-0 polydioxanone (PDS) and covered with pericardial fat pad. Although the postoperative course was uneventful and discharged at postoperative day (POD) nine, bloody sputum appeared and right pneumothorax developed at POD 11. Bronchoscopy revealed a slit-like bronchopleural fistula at intermediate bronchus. By continuous thoracic drainage, the fistula successfully closed at POD 13.

Fluoride Ion Interactions in Alkali-Metal Fluoride-Diol Complexes.

The activity of F- is an important factor in the design of both inorganic and organic reactions involving fluorine compounds. The present study investigates interactions of F- with diols in alkali-metal fluoride-diol complexes. Increases in the reactivities of alkali-metal fluorides and their solubilities in alcohols is observed with increasing cation size. The difference in alkali-metal ion size produces different structural motifs for F--diol complex salts. The CsF complex salt with ethylene glycol (EG), CsF-EG, has a layered structure, whereas the Rb and K complex salts, (RbF)5-(EG)4 and (KF)5-(EG)4, form columnar structures. Comparison of the CsF complex salts with three different diols- EG, 1,3-propylene glycol (PG13), and 1,4-butylene glycol (PG14)-revealed that the diol chain length affects the bridging mode in their layered structures. EG bridges two OH oxygen atoms within the same CsF layer in CsF-EG, whereas PG13 and BG14 bridge two OH oxygen atoms in different CsF layers in (CsF)2-PG13 and CsF-BG14, respectively. The F- ion coordination environment involves interactions between alkali-metal ions and H atom(s) in the diol OH groups, where the F-···H interactions are more dominant than the F-···M+ interaction, based on Hirshfeld surface analyses. The O-H bond weakening observed by infrared spectroscopy also reflects the strengths of the F-···H interactions in these complex salts.

Aggregability of ß(1→4)-linked glucosaminoglucan originating from a sulfur-oxidizing bacterium Thiothrix nivea.

ß-1,4-glucosaminoglucan (GG) was prepared from the sheath of a sulfur-oxidizing bacterium Thiothrix nivea. Recently, GG was found to be adsorbed by cellulose (paper) and is therefore potentially applicable as an aminating agent for cellulose. We attempted to increase the yield of GG using a fed-batch cultivation method. Furthermore, the behavior of GG molecules in water was theoretically and experimentally investigated. NMR analysis in combination with molecular dynamics calculation suggested that GG molecules tend to form soluble aggregates in water. It was experimentally revealed that the self-aggregation is enhanced by the addition of NaCl and reduced temperature. Adsorption of GG onto cellulose via hydrogen bonding was confirmed by molecular dynamics simulation. Adsorption was also promoted in the presence of NaCl but was inhibited by a reduction in temperature. Only 11% of the amino groups in the GG-treated paper was reactive, suggesting that GG molecules adsorbed by the paper were forming aggregates.

Selective alkylation of T-T mismatched DNA using vinyldiaminotriazine-acridine conjugate.

The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T-T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)-acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T-T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T-T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT-acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1.

Structure of a serine-type glutathione S-transferase of Ceriporiopsis subvermispora and identification of the enzymatically important non-canonical residues by functional mutagenesis.

Ceriporiopsis subvermispora (C. subvermispora), one of the white-rot fungi, is known as a selective lignin degrader of the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that are capable of catalyzing the reactions involved in detoxification and metabolic pathways. In this study, a GST of C. subvermispora, named CsGST63524, was overexpressed in E. coli, and then purified by affinity, anion exchange, and size exclusion column chromatography. The crystal structures of the CsGST63524 in ligand-free and complex with GSH were refined at 2.45 and 2.50 Šresolutions, respectively. The sulfur atom of glutathione forms a hydrogen bond with Ser21 of CsGST63524, indicating it is a serine-type GST. Mutagenesis of Ser21 unexpectedly indicated that this serine residue is not essential for the enzymatic activity of CsGST63524. Comparative sequence and structural analyses, together with functional mutagenesis, newly identified the enzymatically important non-canonical amino acid residues, Asn23 and Tyr45, other than the serine residue.

How Does the Recently Discovered Peptide MIP Exhibit Much Higher Binding Affinity than an Anticancer Protein p53 for an Oncoprotein MDM2?

An oncoprotein MDM2 binds to the extreme N-terminal peptide region of a tumor suppressor protein p53 (p53NTD) and inhibits its anticancer activity. We recently discovered a peptide named MIP which exhibits much higher binding affinity for MDM2 than p53NTD. Experiments showed that the binding free energy (BFE) of MDM2-MIP is lower than that of MDM2-p53NTD by approximately -4 kcal/mol. Here, we develop a theoretical method which is successful in reproducing this quantitative difference and elucidating its physical origins. It enables us to decompose the BFE into a variety of energetic and entropic components, evaluate their relative magnitudes, and identify the physical factors driving or opposing the binding. It should be applicable also to the assessment of differences among ligands in the binding affinity for a particular receptor, which is a central issue in modern chemistry. In the MDM2 case, the higher affinity of MIP is ascribed to a larger gain of translational, configurational entropy of water upon binding. This result is useful to the design of a peptide possessing even higher affinity for MDM2 as a reliable drug against a cancer.

Analysis of Interactions between Telomeric i-Motif DNA and a Cyclic Tetraoxazole Compound.

The interaction of a macrocyclic tetraoxazole compound, L2H2-4OTD (1), with two aminoalkyl side chains and telomeric i-motif, was investigated by means of electrophoretic mobility shift assay, circular dichroism spectroscopy, mass spectrometry and NMR spectroscopy analyses. The results indicate that 1 interacts with the i-motif structure at two preferred binding sites.

Identification and characterization of the S-layer formed on the sheath of Thiothrix nivea.

Thiothrix nivea is a filamentous sulfur-oxidizing bacterium common in activated sludge and its filament is covered with a polysaccharide layer called sheath. In this study, we found that T. nivea aggregates under acidic conditions. A hexagonal lattice pattern, a typical morphological feature of proteinaceous S-layers, was newly observed on the surface of the sheath by transmission electron microscopy. The pattern and the acid-dependent aggregation were not observed in T. fructosivorans, a relative sheath-forming bacterium of T. nivea. The putative S-layer of T. nivea was detached by washing with unbuffered tris(hydroxymethyl)aminomethane base (Tris) solution and a protein of 160 kDa was detected by electrophoresis. Based on partial amino acid sequences of the protein, its structural gene was identified. The gene encodes an acidic protein which has a putative secretion signal and a Ca2+-binding domain. The protein was solubilized with urea followed by dialysis in the presence of calcium. A hexagonal lattice pattern was observed in the aggregates formed during dialysis, revealing that the protein is responsible for S-layer formation. Biosorption ability of copper, zinc, and cadmium onto the T. nivea filament decreased upon pretreatment with Tris, demonstrating that the S-layer was involved in metal adsorption. Moreover, aggregation of Escherichia coli was promoted by acidification in the presence of the S-layer protein, suggesting that the protein is potentially applicable as an acid-driven flocculant for other bacteria.

High yield production of fungal manganese peroxidases by E. coli through soluble expression, and examination of the activities.

Oxidative enzymes of white-rot fungi play a key role in lignin biodegradation. Among those fungus, Ceriporiopsis subvermispora degrades lignin before cellulose in wood; C. subvermispora is the only fungus that secretes all known types of manganese peroxidases (CsMnPs). Utilization of lignin-degrading peroxidases has been limited so far due to the lack of efficient preparation methods and intensive characterization. In this study, we developed a highly efficient method to prepare active CsMnPs through soluble expression by E. coli, which had long been impossible. The genes of MnPs selected from each subfamily were codon-optimized and expressed under the control of a cold shock promoter. A proper level of heme incorporation was achieved by continuous addition of hemin during cultivation. As much as 3 mg of purified MnPs was obtained from 100 mL culture, which is an about 20-fold higher yield than that from inclusion bodies through refolding. Further improvement of the solubility on the expression was achieved by combinatorial coexpression of chaperones. All obtained MnPs had heme-to-protein ratios as high as those of native MnPs. They were all active below pH 5. Our method is applicable to other fungal-secreted enzymes should help the progress of their basic characterization and application for better utilization of woody biomass.

Influence of the DNA sequence/length and pH on deaminase activity, as well as the roles of the amino acid residues around the catalytic center of APOBEC3F.

APOBEC3F (A3F), an apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) family protein, catalyzes cytosine-to-uracil conversion in single-stranded (ss) DNA. A3F acts as an inhibitor of retrovirus replication and exhibits antiviral activity against viral infectivity factor (Vif)-deficient human immunodeficiency virus 1 (HIV-1). Previous studies have mostly been focused on the interaction between A3F and Vif, and the studies on A3F's deamination properties are limited. Here, we report comprehensive characterization of the deaminase activity and ssDNA binding of the C-terminal domain (CTD) of A3F. It was shown that the deaminase activity of A3F-CTD is affected by the nucleic acid residues adjacent to the target sequence, TC, and that TTCA/G are the most preferred sequences. A3F-CTD deaminates the target sequence in longer ssDNAs most efficiently. Mutation analysis identified the amino acid residues that are responsible for the deaminase activity and ssDNA binding in the loops surrounding the catalytic center. The functions of these residues were rationally interpreted on the basis of the co-crystal structure of A3A-ssDNA and the known roles of the equivalent amino acid residues found in other A3s. Furthermore, we demonstrated that the deaminase activity of A3F-CTD could be regulated through phosphorylation of a putative site, S216. Finally, A3F-CTD was found to be active in a wide pH range (5.5 to 9.5) with similar activity. Interestingly, the A3F-CTD N214H mutant exhibited a dramatic increase in activity at pH 5.5.

The C-terminal cytidine deaminase domain of APOBEC3G itself undergoes intersegmental transfer for a target search, as revealed by real-time NMR monitoring.

APOBEC3G (A3G), an anti-human immunodeficiency virus 1 factor, deaminates cytidines. We examined deamination of two cytidines located separately on substrate ssDNA by the C-terminal domain (CTD) of A3G using real-time NMR monitoring. The deamination preference between the two cytidines was lost when either the substrate or non-substrate ssDNA concentration increased. When the non-substrate ssDNA concentration increased, the deamination activity first increased, but then decreased. This indicates that even a single domain, A3G-CTD, undergoes intersegmental transfer for a target search.

Mechanism of protein-RNA recognition: analysis based on the statistical mechanics of hydration.

We investigate the RBD1-r(GUAGU) binding as a case study using all-atom models for the biomolecules, molecular models for water, and the currently most reliable statistical-mechanical method. RBD1 is one of the RNA-binding domains of mammalian Musashi1 (Msi1), and r(GUAGU) contains the minimum recognition sequence for Msi1, r(GUAG). We show that the binding is driven by a large gain of configurational entropy of water in the entire system. It is larger than the sum of conformational-entropy losses for RBD1 and r(GUAGU). The decrease in RBD1-r(GUAGU) interaction energy upon binding is largely cancelled out by the increase in the sum of RBD1-water, r(GUAGU)-water, and water-water interaction energies. We refer to this increase as "energetic dehydration". The decrease is larger than the increase for the van der Waals component, whereas the opposite is true for the electrostatic component. We give a novel reason for the empirically known fact that protein residues possessing side chains with positive charges and with flat moieties frequently appear within protein-RNA binding interfaces. A physical picture of the general protein-RNA binding mechanism is then presented. To achieve a sufficiently large water-entropy gain, shape complementarity at the atomic level needs to be constructed by utilizing the stacking and sandwiching of flat moieties (aromatic rings of the protein and nucleobases of RNA) as fundamental motifs. To compensate for electrostatic energetic dehydration, charge complementarity becomes crucial within the binding interface. We argue the reason why the RNA recognition motif (RRM) is the most ubiquitous RNA binding domain.