Femtosecond X-ray Laser Reveals Intact Sea-Island Structures of Metastable Solid-State Electrolytes for Batteries.

Experimental characterization of the nanostructure of metastable functional materials has attracted significant attention with recent advances in computational materials discovery. However, since metastable glass-ceramics are easily damaged by irradiation, damage-free nanoimaging has not been realized thus far. Herein, we propose novel high-contrast coherent diffractive imaging that quantitatively analyzes the intact internal nanostructure of metastable glass-ceramics using femtosecond X-ray pulses. The immersion of sample particles in a solvent helps enhance the reconstructed image contrast and allows us to distinguish an ∼7% electron density difference between an amorphous form and crystals. Furthermore, morphological operations with a band-pass filter quantitatively elucidate the depth information. The evaluated volume ratio of the amorphous to crystalline phases is ∼2.5:1 for the measured metastable (Li2S)70-(P2S5)30 glass-ceramic particle. Sulfide glass-ceramics are used as electrolytes for all-solid-state batteries, which are indispensable for reducing the carbon footprint. Our results will facilitate structural studies on fragile metastable materials with important scientific and industrial implications.

Vibrio cholerae biofilm scaffolding protein RbmA shows an intrinsic, phosphate-dependent autoproteolysis activity.

Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which biofilm communities are considered to be environmental reservoirs. In endemic regions, and after algal blooms, which may result from phosphate enrichment following agricultural runoff, the bacterium is released from biofilms resulting in seasonal disease outbreaks. However, the molecular mechanism by which V. cholerae senses its environment and switches lifestyles from the biofilm-bound state to the planktonic state is largely unknown. Here, we report that the major biofilm scaffolding protein RbmA undergoes autocatalytic proteolysis via a phosphate-dependent induced proximity activation mechanism. Furthermore, we show that RbmA mutants that are defective in autoproteolysis cause V. cholerae biofilms to grow larger and mechanically stronger, correlating well with the observation that RbmA stability directly affects microbial community homeostasis and rheological properties. In conclusion, our biophysical study characterizes a novel phosphate-dependent breakdown pathway of RbmA, while microbiological data suggest a new, sensory role of this biofilm scaffolding element.

Design of a liquid cell toward three-dimensional imaging of unidirectionally-aligned particles in solution using X-ray free-electron lasers.

X-ray free-electron lasers (XFELs) opened up a possibility for molecular-scale single particle imaging (SPI) without the need for crystallization. In SPI experiments, the orientation of each particle has to be determined from the measured diffraction pattern. Preparing unidirectionally-aligned particles can facilitate the determination of the sample orientation. Here, we show the design principles of a liquid cell for three-dimensional imaging of unidirectionally-aligned particles in solution with XFELs. The liquid cell was designed so that neither incident X-rays nor diffracted X-rays are blocked by the substrate of the liquid cell even at high tilt angles. As a feasibility evaluation, we performed coherent diffraction measurements using the cells with a 1 µm focused XFEL beam. We successfully measured coherent diffraction patterns of a nano-fabricated metal pattern at 70° tilt angle and obtained the reconstructed image by applying iterative phase retrieval. The liquid cell will be usefully applied to molecular-scale SPI by using more tightly focused XFELs. In particular, imaging of membrane proteins embedded in lipid membranes is expected to have an enormous impact on life science and medicine.

Human DNA Polymerase µ Can Use a Noncanonical Mechanism for Multiple Mn2+-Mediated Functions.

Recent research on the structure and mechanism of DNA polymerases has continued to generate fundamentally important features, including a noncanonical pathway involving "prebinding" of metal-bound dNTP (MdNTP) in the absence of DNA. While this noncanonical mechanism was shown to be a possible subset for African swine fever DNA polymerase X (Pol X) and human Pol λ, it remains unknown whether it could be the primary pathway for a DNA polymerase. Pol µ is a unique member of the X-family with multiple functions and with unusual Mn2+ preference. Here we report that Pol µ not only prebinds MdNTP in a catalytically active conformation but also exerts a Mn2+ over Mg2+ preference at this early stage of catalysis, for various functions: incorporation of dNTP into a single nucleotide gapped DNA, incorporation of rNTP in the nonhomologous end joining (NHEJ) repair, incorporation of dNTP to an ssDNA, and incorporation of an 8-oxo-dGTP opposite template dA (mismatched) or dC (matched). The structural basis of this noncanonical mechanism and Mn2+ over Mg2+ preference in these functions was analyzed by solving 19 structures of prebinding binary complexes, precatalytic ternary complexes, and product complexes. The results suggest that the noncanonical pathway is functionally relevant for the multiple functions of Pol µ. Overall, this work provides the structural and mechanistic basis for the long-standing puzzle in the Mn2+ preference of Pol µ and expands the landscape of the possible mechanisms of DNA polymerases to include both mechanistic pathways.

Binding and Enhanced Binding between Key Immunity Proteins TRAF6 and TIFA.

Human tumor necrosis factor receptor associated factor (TRAF)-interacting protein, with a forkhead-associated domain (TIFA), is a key regulator of NF-κB activation. It also plays a key role in the activation of innate immunity in response to bacterial infection, through heptose 1,7-bisphosphate (HBP); a metabolite of lipopolysaccharide (LPS). However, the mechanism of TIFA function is largely unexplored, except for the suggestion of interaction with TRAF6. Herein, we provide evidence for direct binding, albeit weak, between TIFA and the TRAF domain of TRAF6, and it is shown that the binding is enhanced for a rationally designed double mutant, TIFA S174Q/M179D. Enhanced binding was also demonstrated for endogenous full-length TRAF6. Furthermore, the structures of the TRAF domain complexes with the consensus TRAF-binding peptides from the C terminus of wild-type and S174Q/M179D mutant TIFA, showing salt-bridge formation between residues 177-181 of TIFA and the binding pocket residues of the TRAF domain, were solved. Taken together, the results provide direct evidence and a structural basis for the TIFA-TRAF6 interaction, and show how this important biological function can be modulated.

Ribosomal protein L31 in Escherichia coli contributes to ribosome subunit association and translation, whereas short L31 cleaved by protease 7 reduces both activities.

Ribosomes routinely prepared from Escherichia coli strain K12 contain intact (70 amino acids) and short (62 amino acids) forms of ribosomal protein L31. By contrast, ribosomes prepared from ompT mutant cells, which lack protease 7, contain only intact L31, suggesting that L31 is cleaved by protease 7 during ribosome preparation. We compared ribosomal subunit association in wild-type and ompT - strains. In sucrose density gradient centrifugation under low Mg2+ , 70S content was very high in ompT - ribosomes, but decreased in the wild-type ribosomes containing short L31. In addition, ribosomes lacking L31 failed to associate ribosomal subunits in low Mg2+ . Therefore, intact L31 is required for subunit association, and the eight C-terminal amino acids contribute to the association function. In vitro translation was assayed using three different systems. Translational activities of ribosomes lacking L31 were 40% lower than those of ompT - ribosomes with one copy of intact L31, indicating that L31 is involved in translation. Moreover, in the stationary phase, L31 was necessary for 100S formation. The strain lacking L31 grew very slowly. A structural analysis predicted that the L31 protein spans the 30S and 50S subunits, consistent with the functions of L31 in 70S association, 100S formation, and translation.

Phospho-Priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation.

The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.

Yolk/Shell Assembly of Gold Nanoparticles by Size Segregation in Solution.

We demonstrate that binary mixtures of small and large gold nanoparticles (GNPs) (5/15, 5/30, 10/30, and 15/30 nm in diameter) in the presence of a glucose-terminated fluorinated oligo(ethylene glycol) ligand can spontaneously form size-segregated assemblies. The outermost layer of the assembly is composed of a single layer of small-sized GNPs, while the larger-sized GNPs are located in the interior, forming what is referred to as a yolk/shell assembly. Time course study reveals that small and large GNPs aggregate together, and these kinetically trapped aggregations were transformed into a size-segregated structure by repeating fusions. A yolk/shell structure was directly visualized in solution by X-ray laser diffraction imaging, indicating that the structure was truly formed in solution, but not through a drying process.

Essentiality of threonylcarbamoyladenosine (t(6)A), a universal tRNA modification, in bacteria.

Threonylcarbamoyladenosine (t(6)A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t(6)A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t(6)A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNA(Ile)-lysidine synthetase. We confirm that t(6)A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t(6)A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t(6)A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t(6)A(-) D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t(6)A in tRNAs. Thus, although t(6)A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.

Crystal structure of the MazG-related nucleoside triphosphate pyrophosphohydrolase from Thermotoga maritima MSB8.

The MazG family proteins, which are highly conserved in bacteria, are nucleoside triphosphate pyrophosphohydrolases that hydrolyze all canonical nucleoside triphosphates, and are also involved in removing noncanonical nucleoside triphosphates to prevent their incorporation into DNA or RNA. The primary structure of TM0360 from Thermotoga maritima MSB8 suggested that TM0360 is a MazG-related nucleoside triphosphate pyrophosphohydrolase. The crystal structure of the TM0360 protein was determined by the MAD technique at 2.0 Å resolution. The asymmetric unit contains an intact dimer molecule. The overall structure of TM0360 is similar to the known structures of the dimeric MazG protein and dUTPases. The putative NTP binding pocket in TM0360, identified by considering the probable NTP-interacting residues and structural features, suggested that TM0360 resembles the C-terminal domain of Escherichia coli MazG, although TM0360 may be a truncated paralog of the N-terminal domain of T. maritima MazG (TM0913), according to its primary structure. The putative function of TM0360 is discussed, based on structural homology.

Synthesis of Janus-like gold nanoparticles with hydrophilic/hydrophobic faces by surface ligand exchange and their self-assemblies in water.

This study aims at the synthesis of Janus gold nanoparticles (Janus GNPs) with hydrophilic/hydrophobic faces by a simple ligand exchange reaction in an homogeneous system and at the elucidation of the self-assembled structures of the Janus GNPs in water. As hydrophilic surface ligands, we synthesized hexaethylene glycol (E6)-terminated thiolate ligands with C3, C7, or C11 alkyl chains, referred to as E6C3, E6C7, and E6C11, respectively. As a hydrophobic ligand, a butyl-headed thiolate ligand C4-E6C11, in which a C4 alkyl was introduced on the E6C11 terminus, was synthesized. The degree of segregation between the two ligands on the GNPs (5 nm in diameter) was examined by matrix-assisted laser desorption/ionization time-of fright mass spectrometry (MALDI-TOF MS) analysis. We found that the choice of immobilization methods, one-step or two-step addition of the two ligands to the GNP solution, crucially affects the degree of segregation. The two-step addition of a hydrophilic ligand (E6C3) followed by a hydrophobic ligand (C4-E6C11) produced a large degree of segregation on the GNPs, providing Janus-like GNPs. When dispersed in water, these Janus-like GNPs formed assemblies of ∼160 nm in diameter, whereas Domain GNPs, in which the two ligands formed partial domains on the surface, were precipitated even when the molar ratio of the hydrophilic ligand and the hydrophobic ligand on the surface of the NPs was almost 1:1. The assembled structure of the Janus-like GNPs in water was directly observed by pulsed coherent X-ray solution scattering using an X-ray free-electron laser, revealing irregular spherical structures with uneven surfaces.

Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes.

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNA(Arg)ICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNA(Arg)CCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA(Arg)CCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNA(Arg) and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA(Arg)ACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNA(Arg)ACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA(Arg)CCG and tadA, and then acquired a new tRNA(Arg)UCG. This permitted further tRNA(Arg)ACG mutations with tRNA(Arg)GCG or its disappearance, leaving a single tRNA(Arg)UCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.

Crystal structure of tRNA m(1)A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-L-methionine.

The N (1)-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m(1)A58 methyltransferase TrmI, using S-adenosyl-L-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.

Conservation of two distinct types of 100S ribosome in bacteria.

In bacteria, 70S ribosomes (consisting of 30S and 50S subunits) dimerize to form 100S ribosomes, which were first discovered in Escherichia coli. Ribosome modulation factor (RMF) and hibernation promoting factor (HPF) mediate this dimerization in stationary phase. The 100S ribosome is translationally inactive, but it dissociates into two translationally active 70S ribosomes after transfer from starvation to fresh medium. Therefore, the 100S ribosome is called the 'hibernating ribosome'. The gene encoding RMF is found widely throughout the Gammaproteobacteria class, but is not present in any other bacteria. In this study, 100S ribosome formation in six species of Gammaproteobacteria and eight species belonging to other bacterial classes was compared. There were several marked differences between the two groups: (i) Formation of 100S ribosomes was mediated by RMF and short HPF in Gammaproteobacteria species, similar to E. coli, whereas it was mediated only by long HPF in the other bacterial species; (ii) RMF/short HPF-mediated 100S ribosome formation occurred specifically in stationary phase, whereas long HPF-mediated 100S ribosome formation occurred in all growth phases; and (iii) 100S ribosomes formed by long HPF were much more stable than those formed by RMF and short HPF.

Characterization and structure of the Aquifex aeolicus protein DUF752: a bacterial tRNA-methyltransferase (MnmC2) functioning without the usually fused oxidase domain (MnmC1).

Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm(5)U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm(5)U34) by the MnmE-MnmG enzyme complex. The cmnm(5)U34 is further modified to mnm(5)U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm(5)U into 5-aminomethyluridine (nm(5)U34), and this reaction is immediately followed by the methylation of the free amino group into mnm(5)U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm(5)U in vitro, to form mnm(5)U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm(5)U34 in tRNAs.

Crystal structure of putative CbiT from Methanocaldococcus jannaschii: an intermediate enzyme activity in cobalamin (vitamin B12) biosynthesis.

BACKGROUND: In the anaerobic pathway of cobalamin (vitamin B12) synthesis, the CbiT enzyme plays two roles, as a cobalt-precorrin-7 C15-methyltransferase and a C12-decarboxylase, to produce the intermediate, cobalt-precorrin 8. RESULTS: The primary structure of the hypothetical protein MJ0391, from Methanocaldococcus jannaschii, suggested that MJ0391 is a putative CbiT. Here, we report the crystal structure of MJ0391, solved by the MAD procedure and refined to final R-factor and R-free values of 19.8 & 27.3%, respectively, at 2.3 Å resolution. The asymmetric unit contains two NCS molecules, and the intact tetramer generated by crystallographic symmetry may be functionally important. The overall tertiary structure and the tetrameric arrangements are highly homologous to those found in MT0146/CbiT from Methanobacterium thermoautotrophicum. CONCLUSIONS: The conservation of functional residues in the binding site for the co-factor, AdoMet, and in the putative precorrin-7 binding pocket suggested that MJ0391 may also possess CbiT activity. The putative function of MJ0391 is discussed, based on structural homology.

Structure of the hypothetical DUF1811-family protein GK0453 from Geobacillus kaustophilus HTA426.

The crystal structure of a conserved hypothetical protein, GK0453, from Geobacillus kaustophilus has been determined to 2.2 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 75.69, c = 64.18 Å. The structure was determined by the molecular-replacement method and was refined to a final R factor of 22.6% (R(free) = 26.3%). Based on structural homology, the GK0453 protein possesses two independent binding sites and hence it may simultaneously interact with two proteins or with a protein and a nucleic acid.

Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR).

Polymerase chain reaction (PCR)-related technologies are hampered mainly by two types of error: nonspecific amplification and DNA polymerase-generated mutations. Here, we report that both errors can be suppressed by the addition of a DNA mismatch-recognizing protein, MutS, from a thermophilic bacterium. Although it had been expected that MutS has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. On the basis of this finding, we propose that MutS binds a mismatched primer-template complex, thereby preventing the approach of DNA polymerase to the 3' end of the primer. Our simple methodology improves the efficiency and accuracy of DNA amplification and should therefore benefit various PCR-based applications, ranging from basic biological research to applied medical science.

Profiling of lipids in Thermus thermophilus HB8 grown under various conditions.

The membrane lipids of Thermus species have unique structures. Only four polar lipid species have so far been identified in Thermus thermophilus HB8; namely, are two phosphoglycolipids and two glycolipids, both of which have three branched fatty acid chains. Other lipid molecules may be present; however, they have not been identified so far. To clarify the whole lipid profile of T. thermophilus HB8, we cultured this organism under four different growth (temperature and/or nutrition) conditions and analyzed the compositions of polar lipids and fatty acids by high-performance thin-layer chromatography (HPTLC) and gas chromatograph-mass spectrometry (GCï½°MS), respectively. Thirty-one lipid spots were detected on HPTLC plates and profiled in terms of the presence or absence of phosphate, amino, and sugar groups. Then, we allocated ID numbers to all the spots. Comparative analyses of these polar lipids showed that the diversity of lipid molecules increased under high temperature and minimal medium conditions. In particular, aminolipid species increased under high temperature conditions. As for the fatty acid comparison by GC-MS, iso-branched even-numbered carbon atoms, which are unusual in this organism, significantly increased under the minimal medium condition, suggesting that kinds of branched amino acids at the fatty acid terminus varies under different nutrition conditions. In this study, several unidentified lipids were detected, and elucidation of the lipid structures will provide important information on the environmental adaptation of bacteria.

Whole genome analyses for c-type cytochromes associated with respiratory chains in the extreme thermophile, Thermus thermophilus.

In thermophilic microorganisms, c-type cytochrome (cyt) proteins mainly function in the respiratory chain as electron carriers. Genome analyses at the beginning of this century revealed a variety of genes harboring the heme c motif. Here, we describe the results of surveying genes with the heme c motif, CxxCH, in a genome database comprising four strains of Thermus thermophilus, including strain HB8, and the confirmation of 19 c-type cytochromes among 27 selected genes. We analyzed the 19 genes, including the expression of four, by a bioinformatics approach to elucidate their individual attributes. One of the approaches included an analysis based on the secondary structure alignment pattern between the heme c motif and the 6th ligand. The predicted structures revealed many cyt c domains with fewer ß-strands, such as mitochondrial cyt c, in addition to the ß-strand unique to Thermus inserted in cyt c domains, as in T. thermophilus cyt c552 and caa3 cyt c oxidase subunit IIc. The surveyed thermophiles harbor potential proteins with a variety of cyt c folds. The gene analyses led to the development of an index for the classification of cyt c domains. Based on these results, we propose names for T. thermophilus genes harboring the cyt c fold.