Nanoparticle Superlattices Driven by Linker-Mediated Covalent Bonding Interaction.

The stability of the nanoparticle superlattice (NPSL) is essential for realizing its broad spectrum of potential applications. Here, we report a linker-mediated covalent bonding interaction method for the synthesis of highly stable NPSLs. Adipic acid is used as a linker molecule which connects two Au NPs functionalized with 6-mercaptohexanol through esterification reactions in the presence of H2SO4. As-prepared NPSLs are mostly fcc Wulff polyhedra with a fairly narrow size distribution and are highly stable in solvents of different polarities and pHs (0-14) as well as in dry conditions and at temperatures as high as 175 °C. The formation of NPSLs involves random homogeneous nucleation simultaneously accompanied by growth, a gradual change of the growth mode from reaction-controlled to diffusion-controlled with time, and the oriented attachments of small crystals. The size of the NPSL can be easily tuned by the concentration of linker molecules and the reaction temperature.

Modulating the folding and binding of peptides using a stimuli-responsive molecular tweezer.

This study presents the development of a ß-hairpin (tryptophan zipper, Trpzip)-based molecular tweezer (MT) that can control the folding and binding of α-helical peptides. When an α-helix isolated from the p53 protein was conjugated with Trpzip in an optimized macrocyclic structure, the folded ß-hairpin stabilized the helix conformation through the side chain-to-side chain stapling strategy, which notably enhanced target (hDM2) affinity of the peptide. On the other hand, the helicity and binding affinity were significantly reduced when the hairpin was unfolded by a redox stimulus. This stimulus-responsive property was translated into the effective capture and release of model multivalent biomaterials, hDM2-gold nanoparticle conjugates. Since numerous protein interactions are mediated by α-helical peptides, these results suggest that the ß-hairpin-based MT holds great potential to be utilized in various biomedical applications, such as protein interaction inhibition and cancer biomarker (e.g., circulating tumor cells and exosomes) detection.

Synchrotron X-ray study of intrinsically disordered and polyampholytic Tau 4RS and 4RL under controlled ionic strength.

Aggregated and hyperphosphorylated Tau is one of the pathological hallmarks of Alzheimer's disease. Tau is a polyampholytic and intrinsically disordered protein (IDP). In this paper, we present for the first time experimental results on the ionic strength dependence of the radius of gyration (Rg) of human Tau 4RS and 4RL isoforms. Synchrotron X-ray scattering revealed that 4RS Rg is regulated from 65.4 to 58.5 Å and 4RL Rg is regulated from 70.9 to 57.9 Å by varying ionic strength from 0.01 to 0.592 M. The Rg of 4RL Tau is larger than 4RS at lower ionic strength. This result provides an insight into the ion-responsive nature of intrinsically disordered and polyampholytic Tau, and can be implicated to the further study of Tau-Tau and Tau-tubulin intermolecular structure in ionic environments.

USP35 dimer prevents its degradation by E3 ligase CHIP through auto-deubiquitinating activity.

Recently, a number of reports on the importance of USP35 in cancer have been published. However, very little is known about the exact mechanism by which USP35 activity is regulated. Here, we show the possible regulation of USP35 activity and the structural specificity affecting its function by analyzing various fragments of USP35. Interestingly, the catalytic domain of USP35 alone does not exhibit deubiquitinating activity; in contrast, the C-terminal domain and insertion region in the catalytic domain is required for full USP35 activity. Additionally, through its C-terminal domain, USP35 forms a homodimer that prevents USP35 degradation. CHIP bound to HSP90 interacts with and ubiquitinates USP35. However, when fully functional USP35 undergoes auto-deubiquitination, which attenuates CHIP-mediated ubiquitination. Finally, USP35 dimer is required for deubiquitination of the substrate Aurora B and regulation of faithful mitotic progression. The properties of USP35 identified in this study are a unique homodimer structure, regulation of deubiquitinating activity through this, and utilization of a novel E3 ligase involved in USP35 auto-deubiquitination, which adds another complexity to the regulation of deubiquitinating enzymes.

Crystal structure of multi-functional enzyme FadB from Cupriavidus necator: Non-formation of FadAB complex.

Cupriavidus necator H16 is a gram-negative chemolithoautotrophic bacterium that has been extensively studied for biosynthesis and biodegradation of polyhydroxyalkanoate (PHA) plastics. To improve our understanding of fatty acid metabolism for PHA production, we determined the crystal structure of multi-functional enoyl-CoA hydratase from Cupriavidus necator H16 (CnFadB). The predicted model of CnFadB created by AlphaFold was used to solve the phase problem during determination of the crystal structure of the protein. The CnFadB structure consists of two distinctive domains, an N-terminal enol-CoA hydratase (ECH) domain and a C-terminal 3-hydroxyacyl-CoA dehydrogenase (HAD) domain, and the substrate- and cofactor-binding modes of these two functional domains were identified. Unlike other known FadB enzymes that exist as dimers complexed with FadA, CnFadB functions as a monomer without forming a complex with CnFadA. Small angle X-ray scattering (SAXS) measurement further proved that CnFadB exists as a monomer in solution. The non-sequential action of FadA and FadB in C. necator appears to affect ß-oxidation and PHA synthesis/degradation.

Structural features of a minimal intact methyltransferase of a type I restriction-modification system.

Type I restriction-modification enzymes are oligomeric proteins composed of methylation (M), DNA sequence-recognition (S), and restriction (R) subunits. The different bipartite DNA sequences of 2-4 consecutive bases are recognized by two discerned target recognition domains (TRDs) located at the two-helix bundle of the two conserved regions (CRs). Two M-subunits and a single S-subunit form an oligomeric protein that functions as a methyltransferase (M2S1 MTase). Here, we present the crystal structure of the intact MTase from Vibrio vulnificus YJ016 in complex with the DNA-mimicking Ocr protein and the S-adenosyl-L-homocysteine (SAH). This MTase includes the M-domain with a helix tail (M-tail helix) and the S1/2-domain of a TRD and a CR α-helix. The Ocr binds to the cleft of the TRD surface and SAH is located in the pocket within the M-domain. The solution- and negative-staining electron microscopy-based reconstructed (M1S1/2)2 structure reveals a symmetric (S1/2)2 assembly using two CR-helices and two M-tail helices as a pivot, which is plausible for recognizing two DNA regions of same sequence. The conformational flexibility of the minimal M1S1/2 MTase dimer indicates a particular state resembling the structure of M2S1 MTases.

Newly Found Digital Memory Characteristics of Pyrrolidone- and Succinimide-Based Polymers.

This study reports for the first time the excellent nonvolatile and volatile digital memory characteristics of polymers bearing 2-pyrrolidone and succinimide moieties. A series of new polymers is synthesized from poly(ethylene-alt-maleic anhydride) and four alcohol derivatives with and without 2-pyrrolidone and succinimide moieties. All polymers, including polyvinylpyrrolidone, are found to be thermally stable up to 195 °C or higher, and characterized regarding their molecular orbital energy levels, bandgap, and resistive digital memory behaviors. Excitingly, the polymers bearing either 2-pyrrolidone or succinimide moiety demonstrate p-type digital memory behaviors with high ON/OFF current ratios and long reliabilities. Nonvolatile digital memory performance is achieved over the film thickness range of 10-80 nm, whereas volatile digital memory is demonstrated over a much narrower range of film thickness. All digital memory performances can be originated from the 2-pyrrolidone and succinimide moieties possessing high affinity and stabilization power to charges via charge traps and transformations based on a hopping conduction process. Hence, these new polymers are suitable for the production of high-performance p-type nonvolatile and volatile digital memory devices. Moreover, 2-pyrrolidone and succinimide can be used as new and economical electroactive building blocks for the development of advanced digital memory materials.

Molecular Analysis of the Interaction between Human PTPN21 and the Oncoprotein E7 from Human Papillomavirus Genotype 18.

Human papillomaviruses (HPVs) cause cellular hyperproliferation-associated abnormalities including cervical cancer. The HPV genome encodes two major viral oncoproteins, E6 and E7, which recruit various host proteins by direct interaction for proteasomal degradation. Recently, we reported the structure of HPV18 E7 conserved region 3 (CR3) bound to the protein tyrosine phosphatase (PTP) domain of PTPN14, a well-defined tumor suppressor, and found that this intermolecular interaction plays a key role in E7-driven transformation and tumorigenesis. In this study, we carried out a molecular analysis of the interaction between CR3 of HPV18 E7 and the PTP domain of PTPN21, a PTP protein that shares high sequence homology with PTPN14 but is putatively oncogenic rather than tumor-suppressive. Through the combined use of biochemical tools, we verified that HPV18 E7 and PTPN21 form a 2:2 complex, with a dissociation constant of 5 nM and a nearly identical binding manner with the HPV18 E7 and PTPN14 complex. Nevertheless, despite the structural similarities, the biological consequences of the E7 interaction were found to differ between the two PTP proteins. Unlike PTPN14, PTPN21 did not appear to be subjected to proteasomal degradation in HPV18-positive HeLa cervical cancer cells. Moreover, knockdown of PTPN21 led to retardation of the migration/invasion of HeLa cells and HPV18 E7-expressing HaCaT keratinocytes, which reflects its protumor activity. In conclusion, the associations of the viral oncoprotein E7 with PTPN14 and PTPN21 are similar at the molecular level but play different physiological roles.

Seventeen-Armed Star Polystyrenes in Various Molecular Weights: Structural Details and Chain Characteristics.

Star-shaped polymers are very attractive because of their potential application ability in various technological areas due to their unique molecular topology. Thus, information on the molecular structure and chain characteristics of star polymers is essential for gaining insights into their properties and finding better applications. In this study, we report molecular structure details and chain characteristics of 17-armed polystyrenes in various molecular weights: 17-Arm(2k)-PS, 17-Arm(6k)-PS, 17-Arm(10k)-PS, and 17-Arm(20k)-PS. Quantitative X-ray scattering analysis using synchrotron radiation sources was conducted for this series of star polymers in two different solvents (cyclohexane and tetrahydrofuran), providing a comprehensive set of three-dimensional structure parameters, including radial density profiles and chain characteristics. Some of the structural parameters were crosschecked by qualitative scattering analysis and dynamic light scattering. They all were found to have ellipsoidal shapes consisting of a core and a fuzzy shell; such ellipse nature is originated from the dendritic core. In particular, the fraction of the fuzzy shell part enabling to store desired chemicals or agents was confirmed to be exceptionally high in cyclohexane, ranging from 74 to 81%; higher-molecular-weight star polymer gives a larger fraction of the fuzzy shell. The largest fraction (81%) of the fuzzy shell was significantly reduced to 52% in tetrahydrofuran; in contrast, the lowest fraction (19%) of core was increased to 48%. These selective shell contraction and core expansion can be useful as a key mechanism in various applications. Overall, the 17-armed polystyrenes of this study are suitable for applications in various technological fields including smart deliveries of drugs, genes, biomedical imaging agents, and other desired chemicals.

Structural insights into the regulation of SigB activity by RsbV and RsbW.

Bacillus subtilis SigB is an alternative sigma factor that initiates the transcription of stress-responsive genes. The anti-sigma factor RsbW tightly binds SigB to suppress its activity under normal growth conditions and releases it when nonphosphorylated RsbV binds to RsbW in response to stress signals. To understand the regulation of SigB activity by RsbV and RsbW based on structural features, crystal structures and a small-angle X-ray scattering (SAXS) envelope structure of the RsbV-RsbW complex were determined. The crystal structures showed that RsbV and RsbW form a heterotetramer in a similar manner to a SpoIIAA-SpoIIAB tetramer. Multi-angle light scattering and SAXS revealed that the RsbV-RsbW complex is an octamer in solution. Superimposition of the crystal structure on the SAXS envelope structure showed that the unique dimeric interface of RsbW mediates the formation of an RsbV-RsbW octamer and does not prevent RsbV and SigB from binding to RsbW. These results provide structural insights into the molecular assembly of the RsbV-RsbW complex and the regulation of SigB activity.

Mechanism for Transition of Reverse Cylindrical Micelles to Spherical Micelles Induced by Diverse Alcohols.

Herein, the effects of various alcohols on lecithin/CaCl2 organogels are investigated. Mixtures of lecithin and CaCl2 form reverse cylindrical micelles, resulting in optically transparent organogels. The addition of various alcohols to a mixture of lecithin and CaCl2 induces a decrease in viscosity through which reverse cylindrical micelles are transformed into spherical micelles (or short cylindrical micelles). Long-hydrocarbon-chain alcohols decrease the viscosity of lecithin/CaCl2 mixtures more efficiently. Hydrogen bonding and hydrocarbon chain interactions between lecithin and alcohol play important roles in the morphological transition. More importantly, isothermal titration calorimetry was conducted to obtain thermodynamic variables such as the enthalpy, equilibrium constant, Gibbs free energy, entropy, and stoichiometry of the associated molecules observed in the transition. It was found that the transition is an entropically driven process, in which the endothermic and exothermic behaviors were observed depending on the hydrocarbon chain length in the alcohol. In addition, the enthalpy for the association of the alcohol with lecithin showed a linear relationship depending on the hydrocarbon chain length, in which the magnitude of hydrogen bonding and hydrocarbon chain interactions was obtained quantitatively. To the best of our knowledge, this is the first study reporting the thermodynamic properties of the morphological transition observed in a reverse self-assembly process.

Quantitative Structural Analysis of Polystyrene Nanoparticles Using Synchrotron X-Ray Scattering and Dynamic Light Scattering.

A series of polystyrene nanoparticles (PS-1, PS-2, PS-3, and PS-4) in aqueous solutions were investigated in terms of morphological structure, size, and size distribution. Synchrotron small-angle X-ray scattering analysis (SAXS) was carried out, providing morphology details, size and size distribution on the particles. PS-1, PS-2, and PS-3 were confirmed to behave two-phase (core and shell) spherical shapes, whereas PS-4 exhibited a single-phase spherical shape. They all revealed very narrow unimodal size distributions. The structural parameter details including radial density profile were determined. In addition, the presence of surfactant molecules and their assemblies were detected for all particle solutions, which could originate from their surfactant-assisted emulsion polymerizations. In addition, dynamic light scattering (DLS) analysis was performed, finding only meaningful hydrodynamic size and intensity-weighted mean size information on the individual PS solutions because of the particles' spherical nature. In contrast, the size distributions were extracted unrealistically too broad, and the volume- and number-weighted mean sizes were too small, therefore inappropriate to describe the particle systems. Furthermore, the DLS analysis could not detect completely the surfactant and their assemblies present in the particle solutions. Overall, the quantitative SAXS analysis confirmed that the individual PS particle systems were successfully prepared with spherical shape in a very narrow unimodal size distribution.

Structural insight into bi-functional malonyl-CoA reductase.

The bi-functional malonyl-CoA reductase is a key enzyme of the 3-hydroxypropionate bi-cycle for bacterial CO2 fixation, catalysing the reduction of malonyl-CoA to malonate semialdehyde and further reduction to 3-hydroxypropionate. Here, we report the crystal structure and the full-length architecture of malonyl-CoA reductase from Porphyrobacter dokdonensis. The malonyl-CoA reductase monomer of 1230 amino acids consists of four tandemly arranged short-chain dehydrogenases/reductases, with two catalytic and two non-catalytic short-chain dehydrogenases/reductases, and forms a homodimer through paring contact of two malonyl-CoA reductase monomers. The complex structures with its cofactors and substrates revealed that the malonyl-CoA substrate site is formed by the cooperation of two short-chain dehydrogenases/reductases and one novel extra domain, while only one catalytic short-chain dehydrogenase/reductase contributes to the formation of the malonic semialdehyde-binding site. The phylogenetic and structural analyses also suggest that the bacterial bi-functional malonyl-CoA has a structural origin that is completely different from the archaeal mono-functional malonyl-CoA and malonic semialdehyde reductase, and thereby constitute an efficient enzyme.

Chemically Denatured Structures of Porcine Pepsin using Small-Angle X-ray Scattering.

Porcine pepsin is a gastric aspartic proteinase that reportedly plays a pivotal role in the digestive process of many vertebrates. We have investigated the three-dimensional (3D) structure and conformational transition of porcine pepsin in solution over a wide range of denaturant urea concentrations (0-10 M) using Raman spectroscopy and small-angle X-ray scattering. Furthermore, 3D GASBOR ab initio structural models, which provide an adequate conformational description of pepsin under varying denatured conditions, were successfully constructed. It was shown that pepsin molecules retain native conformation at 0-5 M urea, undergo partial denaturation at 6 M urea, and display a strongly unfolded conformation at 7-10 M urea. According to the resulting GASBOR solution models, we identified an intermediate pepsin conformation that was dominant during the early stage of denaturation. We believe that the structural evidence presented here provides useful insights into the relationship between enzymatic activity and conformation of porcine pepsin at different states of denaturation.

Symmetric Assembly of a Decameric Subcomplex in Human Multi-tRNA Synthetase Complex Via Interactions between Glutathione Transferase-Homology Domains and Aspartyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (AARSs) ligate amino acids to their cognate tRNAs during protein synthesis. In humans, eight AARSs and three non-enzymatic AARS-interacting multifunctional proteins (AIMP1-3), which are involved in various biological processes, form a multi-tRNA synthetase complex (MSC). Elucidation of the structures and multiple functions of individual AARSs and AIMPs has aided current understanding of the structural arrangement of MSC components and their assembly processes. Here, we report the crystal structure of a complex comprising a motif from aspartyl-tRNA synthetase (DRS) and the glutathione transferase (GST)-homology domains of methionyl-tRNA synthetase (MRS), glutamyl-prolyl-tRNA synthetase (EPRS), AIMP2, and AIMP3. In the crystal structure, the four GST domains are assembled in the order of MRS-AIMP3-EPRS-AIMP2, and the GST domain of AIMP2 binds DRS through the ß-sheet in the GST domain. The C-terminus of AIMP3 enhances the binding of DRS to the tetrameric GST complex. A DRS dimer and two GST tetramers binding to the dimer with 2-fold symmetry complete a decameric complex. The formation of this complex enhances the stability of DRS and enables it to retain its reaction intermediate, aspartyl adenylate. Since the catalytic domains of MRS and EPRS are connected to the decameric complex through their flexible linker peptides, and lysyl-tRNA synthetase and AIMP1 are also linked to the complex via the N-terminal region of AIMP2, the DRS-GST tetramer complex functions as a frame in the MSC.

Structural and Molecular Basis for Katanin-Mediated Severing of Glutamylated Microtubules.

Katanin was the first microtubule (MT)-severing enzyme discovered, but how katanin executes MT severing remains poorly understood. Here, we report X-ray crystal structures of the apo and ATPγS-bound states of the catalytic AAA domain of human katanin p60 at 3.0 and 2.9 Å resolution, respectively. Comparison of the two structures reveals conformational changes induced by ATP binding and how such changes ensure hexamer stability. Moreover, we uncover structural details of pore loops (PLs) and show that Arg283, a residue unique to katanin among MT-severing enzymes, protrudes from PL1 and lines the entry of the catalytic pore. Functional studies suggest that PL1 and Arg283 play essential roles in the recognition and remodeling of the glutamylated, C-terminal tubulin tail and regulation of axon growth. In addition, domain-swapping experiments in katanin and spastin suggest that the non-homologous N-terminal region, which contains the MT-interacting and trafficking domain and a linker, confers specificity to the severing process.

Micelle-Assisted Formation of Nanoparticle Superlattices and Thermally Reversible Symmetry Transitions.

Nanoparticle superlattices (NPSLs) are of great interest as materials with designed emerging properties depending on the lattice symmetry as well as composition. The symmetry transition of NPSLs depending on environmental conditions can be an excellent ground for making new stimuli-responsive functional materials. Here, we report a spherical micelle-assisted method to form exceptionally ordered NPSLs which are inherently sensitive to environmental conditions. Upon mixing functionalized gold nanoparticles (AuNPs) with a nonionic surfactant spherical micellar solution, NPSLs of different symmetries such as NaZn13, MgZn2, and AlB2-type are formed depending on the size ratio between micelles and functionalized AuNPs and composition. The NPSLs formed by the spherical micelle-assisted method show thermally reversible order-order (NaZn13-AlB2) and order-disorder (MgZn2-isotropic) symmetry transitions, which are consistent with the Gibbs free energy calculations for binary hard-sphere model. This approach may open up new possibilities for NPSLs as stimuli-responsive functional materials.

Bacillus licheniformis α-amylase: Structural feature in a biomimetic solution and structural changes in extrinsic conditions.

Bacillus licheniformis α-amylase (BLA) in a biomimetic buffer and extrinsic solutions (various pH values, temperatures, and metal ions) has been investigated for the first time in the view of three-dimensional (3D) structure by synchrotron X-ray and dynamic light scattering analyses. BLA in buffer is determined to have a structure resembling its crystallographic structure; but the 3D structure is slightly larger than the crystal structure. Such a structure is maintained with little variations in extrinsic solutions of pH 4.0-9.7, temperature 4-55 °C, and metal ions such as Ba2+, Mg2+, and Li+. These results collectively inform that BLA tends to favorably form a stable monomeric structure, which could provide structural clues to its enzymatic activities in moderate levels. Interestingly, BLA is found to reveal highly expanded structures at 65-75 °C and in Co2+ solution, which could correlate to the significantly pronounced enzymatic activities. However, BLA shows somewhat shrunken structures at pH 3.0 and in Hg2+ solution, supporting for the suppressed activities under these conditions.

Characterization of a Dimeric Arginase From Zymomonas mobilis ZM4.

Many organisms have genes to protect themselves from toxic conditions such as high ethanol and/or ammonia concentrations. When a high ethanol condition is induced to Zymomonas mobilis ZM4, a representative ethanologenic organism, this bacterium overexpresses several genes to overcome this ethanol stress. Among them, we characterized a gene product annotated as an arginase (zmARG) from Z. mobilis ZM4. Even though all of the arginase-determining sequence motifs are not strictly conserved in zmARG, this enzyme converts L-arginine to urea and L-ornithine in the presence of a divalent manganese ion. The revealed high-resolution crystal structure of zmARG shows that it has a typical globular α/ß arginase fold with a protruded C-terminal helix. Two zinc ions reside in the active site, where one metal ion is penta-coordinated and the other has six ligands, discerning this zmARG from the reported arginases with two hexa-liganded metal ions. zmARG forms a dimeric structure in solution as well as in the crystalline state. The dimeric assembly of zmARG is formed mainly by interaction formed between the C-terminal α-helix of one molecule and the α/ß hydrolase fold of another molecule. The presented findings demonstrate the first reported dimeric arginase formed by the C-terminal tail and has two metal ions coordinated by different number of ligands.

Structural Insights into the FtsQ/FtsB/FtsL Complex, a Key Component of the Divisome.

Bacterial cell division is a fundamental process that results in the physical separation of a mother cell into two daughter cells and involves a set of proteins known as the divisome. Among them, the FtsQ/FtsB/FtsL complex was known as a scaffold protein complex, but its overall structure and exact function is not precisely known. In this study, we have determined the crystal structure of the periplasmic domain of FtsQ in complex with the C-terminal fragment of FtsB, and showed that the C-terminal region of FtsB is a key binding region of FtsQ via mutational analysis in vitro and in vivo. We also obtained the solution structure of the periplasmic FtsQ/FtsB/FtsL complex by small angle X-ray scattering (SAXS), which reveals its structural organization. Interestingly, the SAXS and analytical gel filtration data showed that the FtsQ/FtsB/FtsL complex forms a 2:2:2 heterohexameric assembly in solution with the "Y" shape. Based on the model, the N-terminal directions of FtsQ and the FtsB/FtsL complex should be opposite, suggesting that the Y-shaped FtsQ/FtsB/FtsL complex might fit well into the curved membrane for membrane anchoring.