Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum.

Among the epimerases specific to alginate, some of them in Azotobacter genera convert ß-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.

Chemo-mechanical forces modulate the topology dynamics of mesoscale DNA assemblies.

The intrinsic complexity of many mesoscale (10-100 nm) cellular machineries makes it challenging to elucidate their topological arrangement and transition dynamics. Here, we exploit DNA origami nanospring as a model system to demonstrate that tens of piconewton linear force can modulate higher-order conformation dynamics of mesoscale molecular assemblies. By switching between two chemical structures (i.e., duplex and tetraplex DNA) in the junctions of adjacent origami modules, the corresponding stretching or compressing chemo-mechanical stress reversibly flips the backbone orientations of the DNA nanosprings. Both coarse-grained molecular dynamics simulations and atomic force microscopy measurements reveal that such a backbone conformational switch does not alter the right-handed chirality of the nanospring helix. This result suggests that mesoscale helical handedness may be governed by the torque, rather than the achiral orientation, of nanospring backbones. It offers a topology-based caging/uncaging concept to present chemicals in response to environmental cues in solution.

Giant Carbon Nano-Test Tubes as Versatile Imaging Vessels for High-Resolution and In Situ Observation of Proteins.

Cryogenic electron microscopy is one of the fastest and most robust methods for capturing high-resolution images of proteins, but stringent sample preparation, imaging conditions, and in situ radiation damage inflicted during data acquisition directly affect the resolution and ability to capture dynamic details, thereby limiting its broader utilization and adoption for protein studies. We addressed these drawbacks by introducing synthesized giant carbon nano-test tubes (GCNTTs) as radiation-insulating materials that lessen the irradiation impact on the protein during data acquisition, physical molecular concentrators that localize the proteins within a nanoscale field of view, and vessels that create a microenvironment for solution-phase imaging. High-resolution electron microscopy images of single and aggregated hemoglobin molecules within GCNTTs in both solid and solution states were acquired. Subsequent scanning transmission electron microscopy, small-angle neutron scattering, and fluorescence studies demonstrated that the GCNTT vessel protected the hemoglobin molecules from electron irradiation-, light-, or heat-induced denaturation. To demonstrate the robustness of GCNTT as an imaging platform that could potentially augment the study of proteins, we demonstrated the robustness of the GCNTT technique to image an alternative protein, d-fructose dehydrogenase, after cyclic voltammetry experiments to review encapsulation and binding insights. Given the simplicity of the material synthesis, sample preparation, and imaging technique, GCNTT is a promising imaging companion for high-resolution, single, and dynamic protein studies under electron microscopy.

Lipid bilayer-assisted dynamic self-assembly of hexagonal DNA origami blocks into monolayer crystalline structures with designed geometries.

The DNA origami technique is used to construct custom-shaped nanostructures that can be used as components of two-dimensional crystalline structures with user-defined structural patterns. Here, we designed an Mg2+-responsive hexagonal 3D DNA origami block with self-shape-complementary ruggedness on the sides. Hexagonal DNA origami blocks were electrostatically adsorbed onto a fluidic lipid bilayer membrane surface to ensure lateral diffusion. A subsequent increase in the Mg2+ concentration in the surrounding environment induced the self-assembly of the origami blocks into lattices with prescribed geometries based on a self-complementary shape fit. High-speed atomic force microscopy (HS-AFM) images revealed dynamic events involved in the self-assembly process, including edge reorganization, defect splitting, diffusion, and filling, which provide a glimpse into how the lattice structures are self-improved.

Rapid Alkene-Alkene Photo-Cross-Linking on the Base-Flipping-Out Field in Duplex DNA.

Specific chemical reactions by enzymes acting on a nucleobase are realized by flipping the target base out of the helix. Similarly, artificial oligodeoxynucleotides (ODNs) can also induce the base flipping and a specific chemical reaction. We now report an easily prepared and unique structure-providing photo-cross-linking reaction by taking advantage of the base-flipping-out field formed by alkene-type base-flipping-inducing artificial bases. Two 3-arylethenyl-5-methyl-2-pyridone nucleosides with the Ph or An group were synthesized and incorporated into the ODNs. We found that the two Ph derivatives provided the cross-linked product in a high yield only by a 10 s photoirradiation when their alkenes overlap each other in the duplex DNA. The highly efficient reaction enabled forming a cross-linked product even when using the duplex with a low Tm value.

Testing mechanisms of DNA sliding by architectural DNA-binding proteins: dynamics of single wild-type and mutant protein molecules in vitro and in vivo.

Architectural DNA-binding proteins (ADBPs) are abundant constituents of eukaryotic or bacterial chromosomes that bind DNA promiscuously and function in diverse DNA reactions. They generate large conformational changes in DNA upon binding yet can slide along DNA when searching for functional binding sites. Here we investigate the mechanism by which ADBPs diffuse on DNA by single-molecule analyses of mutant proteins rationally chosen to distinguish between rotation-coupled diffusion and DNA surface sliding after transient unbinding from the groove(s). The properties of yeast Nhp6A mutant proteins, combined with molecular dynamics simulations, suggest Nhp6A switches between two binding modes: a static state, in which the HMGB domain is bound within the minor groove with the DNA highly bent, and a mobile state, where the protein is traveling along the DNA surface by means of its flexible N-terminal basic arm. The behaviors of Fis mutants, a bacterial nucleoid-associated helix-turn-helix dimer, are best explained by mobile proteins unbinding from the major groove and diffusing along the DNA surface. Nhp6A, Fis, and bacterial HU are all near exclusively associated with the chromosome, as packaged within the bacterial nucleoid, and can be modeled by three diffusion modes where HU exhibits the fastest and Fis the slowest diffusion.

The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA.

DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

Rational design using sequence information only produces a peptide that binds to the intrinsically disordered region of p53.

Intrinsically disordered regions (IDRs) of proteins are involved in many diseases. The rational drug design against disease-mediating proteins is often based on the 3D structure; however, the flexible structure of IDRs hinders the use of such structure-based design methods. Here, we developed a rational design method to obtain a peptide that can bind an IDR using only sequence information based on the statistical contact energy of amino acid pairs. We applied the method to the disordered C-terminal domain of the tumor suppressor p53. Titration experiments revealed that one of the designed peptides, DP6, has a druggable affinity of ~1 µM to the p53 C-terminal domain. NMR spectroscopy and molecular dynamics simulation revealed that DP6 selectively binds to the vicinity of the target sequence in the C-terminal domain of p53. DP6 inhibits the nonspecific DNA binding of a tetrameric form of the p53 C-terminal domain, but does not significantly affect the specific DNA binding of a tetrameric form of the p53 core domain. Single-molecule measurements revealed that DP6 retards the 1D sliding of p53 along DNA, implying modulation of the target searching of p53. Statistical potential-based design may be useful in designing peptides that target IDRs for therapeutic purposes.

Alkyne-Alkyne Photo-cross-linking on the Flipping-out Field.

The base flip-inducing nucleic acids are expected to create a specific field for various chemical reactions. We now report a novel type of base-flip-inducing oligodeoxynucleotide and photo-cross-linking reaction. Two 3-arylethynyl-5-methyl-2-pyridone nucleosides, Ph and An, were synthesized, and their properties were investigated. The alkyne-alkyne photo-cross-linking rapidly proceeded by taking advantage of the base-flipping-out field where two alkynes overlap each other. This photo-cross-linking would be a new candidate to form cross-linked DNAs.

High Free-Energy Barrier of 1D Diffusion Along DNA by Architectural DNA-Binding Proteins.

Architectural DNA-binding proteins function to regulate diverse DNA reactions and have the defining property of significantly changing DNA conformation. Although the 1D movement along DNA by other types of DNA-binding proteins has been visualized, the mobility of architectural DNA-binding proteins on DNA remains unknown. Here, we applied single-molecule fluorescence imaging on arrays of extended DNA molecules to probe the binding dynamics of three structurally distinct architectural DNA-binding proteins: Nhp6A, HU, and Fis. Each of these proteins was observed to move along DNA, and the salt concentration independence of the 1D diffusion implies sliding with continuous contact to DNA. Nhp6A and HU exhibit a single sliding mode, whereas Fis exhibits two sliding modes. Based on comparison of the diffusion coefficients and sizes of many DNA binding proteins, the architectural proteins are categorized into a new group distinguished by an unusually high free-energy barrier for 1D diffusion. The higher free-energy barrier for 1D diffusion by architectural proteins can be attributed to the large DNA conformational changes that accompany binding and impede rotation-coupled movement along the DNA grooves.

One-Dimensional Search Dynamics of Tumor Suppressor p53 Regulated by a Disordered C-Terminal Domain.

Tumor suppressor p53 slides along DNA and finds its target sequence in drastically different and changing cellular conditions. To elucidate how p53 maintains efficient target search at different concentrations of divalent cations such as Ca2+ and Mg2+, we prepared two mutants of p53, each possessing one of its two DNA-binding domains, the CoreTet mutant having the structured core domain plus the tetramerization (Tet) domain, and the TetCT mutant having Tet plus the disordered C-terminal domain. We investigated their equilibrium and kinetic dissociation from DNA and search dynamics along DNA at various [Mg2+]. Although binding of CoreTet to DNA becomes markedly weaker at higher [Mg2+], binding of TetCT depends slightly on [Mg2+]. Single-molecule fluorescence measurements revealed that the one-dimensional diffusion of CoreTet along DNA consists of fast and slow search modes, the ratio of which depends strongly on [Mg2+]. In contrast, diffusion of TetCT consisted of only the fast mode. The disordered C-terminal domain can associate with DNA irrespective of [Mg2+], and can maintain an equilibrium balance of the two search modes and the p53 search distance. These results suggest that p53 modulates the quaternary structure of the complex between p53 and DNA under different [Mg2+] and that it maintains the target search along DNA.

Gravitropism in leaves of Arabidopsis thaliana (L.) Heynh.

In higher plants, stems and roots show negative and positive gravitropism, respectively. However, current knowledge on the graviresponse of leaves is lacking. In this study, we analyzed the positioning and movement of rosette leaves of Arabidopsis thaliana under light and dark conditions. We found that the radial positioning of rosette leaves was not affected by the direction of gravity under continuous white light. In contrast, when plants were shifted to darkness, the leaves moved upwards, suggesting negative gravitropism. Analysis of the phosphoglucomutase and shoot gravitropism 2-1 mutants revealed that the sedimenting amyloplasts in the leaf petiole are important for gravity perception, as is the case in stems and roots. In addition, our detailed physiological analyses revealed a unique feature of leaf movement after the shift to darkness, i.e. movement could be divided into negative gravitropism and nastic movement. The orientation of rosette leaves is ascribed to a combination of these movements.

In vitro and in vivo inhibitory effects of disodium cromoglycate on influenza virus infection.

Disodium cromoglycate (DSCG) is one of the safest drugs for the prevention of bronchial asthma and allergic rhinitis attacks. The effect of DSCG on acute upper respiratory tract viral infection is still controversial. Here we investigated DSCG inhibition of influenza virus infection in vivo and in vitro. In vivo effects of DSCG on viral infection were assessed using a murine model of respiratory tract infection. Intranasal administration of DSCG protected mice from death induced by infection with influenza virus A/PR/8/34. We analyzed DSCG anti-viral effects in vitro by either (i) treating cells prior to viral adsorption, (ii) treating cells concurrently with viral adsorption, or (iii) treating cells after viral adsorption. DSCG treatment of cells during or after, but not before, viral adsorption significantly inhibited influenza viral infection, indicating DSCG acts on events late in viral infection. DSCG exerts anti-influenza effect both in vitro and in vivo at the doses compatible with treatment for asthma. DSCG marginally inhibited influenza viral neuraminidase and membrane fusion functions, suggesting that DSCG inhibition of viral neuraminidase and fusion activities may partially mediate this anti-influenza effect. Our results indicate that treatment of patients including children with DSCG may take advantages for prevention from influenza virus infection.