Masatada Tamakoshi

Crystal structure of a central stalk subunit C and reversible association/dissociation of vacuole-type ATPase.

The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-Å resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments. The result shows that V-ATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the V1 (soluble) and the Vo (membrane bound) domains. Subunit C, attached to the Vo domain, seems to have a socket like function in attaching the central-stalk subunits of the V1 domain. This architecture seems essential for the reversible association/dissociation of the V1 and the Vo domains, unique for V-ATPase activity regulation.

Molecular Mechanism of Energy Conservation in Polysulfide Respiration.

Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone-coupled reduction of polysulfide, a process important in extreme environments such as deep-sea vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4-Å resolution, revealing how the PsrA subunit recognizes and reduces its unique polyanionic substrate. The integral membrane subunit PsrC was characterized using the natural substrate menaquinone-7 and inhibitors, providing a comprehensive representation of a quinone binding site and revealing the presence of a water-filled cavity connecting the quinone binding site on the periplasmic side to the cytoplasm. These results suggest that polysulfide reductase could be a key energy-conserving enzyme of the T. thermophilus respiratory chain, using polysulfide as the terminal electron acceptor and pumping protons across the membrane via a previously unknown mechanism.

Transcription profile of Thermus thermophilus CRISPR systems after phage infection.

The clustered regularly interspaced short palindromic repeat (CRISPR) systems composed of DNA direct repeats designated as CRISPRs and several CRISPR-associated (cas) genes, which are present in many prokaryotic genomes, make up a host defense system against invading foreign replicons such as phages. In order to investigate the altered expression profiles of the systems after phage infection using a model organism, Thermus thermophilus HB8, which has 12 CRISPR loci, genome-wide transcription profiling of the strain infected with lytic phage PhiYS40 was performed by DNA microarray analysis. Significant alteration of overall mRNA expression gradually increased during infection (i.e., from the eclipse period to the period of host cell lysis). Interestingly, the expression of most cAMP receptor protein (CRP)-regulated genes, including two CRISPR-associated (cas) operons, was most markedly up-regulated, especially around the beginning of host cell lysis, although up-regulation of the crp gene was not observed. The expression of the CRP-regulated genes was less up-regulated in a crp-deficient strain than in the wild type. Thus, it is suggested that cAMP is a signaling molecule that transmits information on phage infection to CRP to up-regulate these genes. On the other hand, the expression of several cas genes and that of CRISPRs were up-regulated independent of CRP, suggesting the involvement of unidentified regulatory factor(s) induced by phage infection. On analysis of the expression profile of the entire genome, we could speculate that upon phage infection, the signal was transmitted to the cells, with host response systems including CRISPR defense systems being activated, while the overall efficiencies of transcription, translation, and metabolism in the cells decreased. These findings will facilitate understanding of the host response mechanism following phage infection.

Imaging live cell in micro-liquid enclosure by X-ray laser diffraction

Emerging X-ray free-electron lasers with femtosecond pulse duration enable single-shot snapshot imaging almost free from sample damage by outrunning major radiation damage processes. In bioimaging, it is essential to keep the sample close to its natural state. Conventional high-resolution imaging, however, suffers from severe radiation damage that hinders live cell imaging. Here we present a method for capturing snapshots of live cells kept in a micro-liquid enclosure array by X-ray laser diffraction. We place living Microbacterium lacticum cells in an enclosure array and successively expose each enclosure to a single X-ray laser pulse from the SPring-8 Angstrom Compact Free-Electron Laser. The enclosure itself works as a guard slit and allows us to record a coherent diffraction pattern from a weakly-scattering submicrometre-sized cell with a clear fringe extending up to a 28-nm full-period resolution. The reconstructed image reveals living whole-cell structures without any staining, which helps advance understanding of intracellular phenomena.

Dodecamer rotor ring defines H+/ATP ratio for ATP synthesis of prokaryotic V-ATPase from Thermus thermophilus

ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) >110 mV, which is composed of a difference in proton concentration (ΔpH) and the electrical potential differences (ΔΨ) across membranes. The optimum rate of synthesis reached 85 s−1, and the H+/ATP ratio of 4.0 ± 0.1 was obtained. ATP was synthesized at a considerable rate solely by ΔpH, indicating ΔΨ was not absolutely required for synthesis. Consistent with the H+/ATP ratio, cryoelectron micrograph images of 2D crystals of the membrane-bound rotor ring of the V-ATPase at 7.0-Å resolution showed the presence of 12 Vo-c subunits, each composed of two transmembrane helices. These results indicate that symmetry mismatch between the rotor and catalytic domains is not obligatory for rotary ATPases/synthases.

ATP Hydrolysis and Synthesis of a Rotary Motor V-ATPase from Thermus thermophilus

Vacuolar-type H+-ATPase (V-ATPase) catalyzes ATP synthesis and hydrolysis coupled with proton translocation across membranes via a rotary motor mechanism. Here we report biochemical and biophysical catalytic properties of V-ATPase from Thermus thermophilus. ATP hydrolysis of V-ATPase was severely inhibited by entrapment of Mg-ADP in the catalytic site. In contrast, the enzyme was very active for ATP synthesis (∼70 s–1) with the Km values for ADP and phosphate being 4.7 ± 0.5 and 460 ± 30 μm, respectively. Single molecule observation showed V-ATPase rotated in a 120° stepwise manner, and analysis of dwelling time allowed the binding rate constant kon for ATP to be estimated (∼1.1 × 106 m–1 s–1), which was much lower than the kon (= Vmax/Km) for ADP (∼1.4 × 107 m–1 s–1). The slower \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{\mathrm{on}}^{\mathrm{ATP}}\) \end{document} than \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(k_{\mathrm{on}}^{\mathrm{ADP}}\) \end{document} and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses.

Structure of a central stalk subunit F of prokaryotic V-type ATPase/synthase from Thermus thermophilus

The crystal structure of subunit F of vacuole‐type ATPase/synthase (prokaryotic V‐ATPase) was determined to of 2.2 Å resolution. The subunit reveals unexpected structural similarity to the response regulator proteins that include the Escherichia coli chemotaxis response regulator CheY. The structure was successfully placed into the low‐resolution EM structure of the prokaryotic holo‐V‐ATPase at a location indicated by the results of crosslinking experiments. The crystal structure, together with the single‐molecule analysis using fluorescence resonance energy transfer, showed that the subunit F exhibits two conformations, a ‘retracted’ form in the absence and an ‘extended’ form in the presence of ATP. Our results postulated that the subunit F is a regulatory subunit in the V‐ATPase.

Screening of stable proteins in an extreme thermophile, Thermus thermophilus

The leuB gene codes for 3‐isopropylmalate dehydrogenase of the leucine biosynthetic pathway in an extreme thermophile, Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature‐sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucine was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.

Conserved Bases in the TΨC Loop of tRNA Are Determinants for Thermophile-specific 2-Thiouridylation at Position 54

2-Thioribothymidine (s2T) is a post-transcriptionally modified nucleoside of U54 specifically found in thermophilic bacterial tRNAs. The 2-thiocarbonyl group of s2T54 is known to be responsible for the thermostability of tRNA. The s2T54 content in tRNA varies depending on the cultivation temperature, a feature that confers thermal adaptation of protein synthesis in Thermus thermophilus. Little is known about the biosynthesis of s2T, including the sulfur donor, modification enzyme, and the tRNA structural requirements. To characterize 2-thiolation at position 54 in tRNA, we constructed an in vivo expression system using tRNAAsp with an altered sequence and a host-vector for T. thermophilus. We were able to detectin vivo activity of s2T54 thiolase using phenyl mercuric gel electrophoresis followed by Northern hybridization. 2-Thiolation at position 54 was identified in the precursor form of the tRNA, indicating that 2-thiolation precedes tRNA processing. To ascertain the elements that determine 2-thiolation in tRNA, systematic site-directed mutagenesis was carried out using the tRNAAspgene. Conserved residues C56 and A58 were identified as major determinants of 2-thiolation, whereas tertiary interaction between the T and D loops and non-conserved nucleosides in the T loop were revealed not to be important for the reaction.

Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus

A leuB strain of Thermus thermophilus, TTY1, was transformed with a plasmid vector that directed expression of 3-isopropylmalate dehydrogenase (IPMDH) of Saccharomyces cerevisiae encoded by the LEU2 gene. The original strain could not grow at 50°C without leucine, probably because of the low stability of S. cerevisiae IPMDH. The mutants that could grow without leucine were selected at 50°, 60°, 62°, 65°, 67°, and 70°C, step by step. All the mutant strains except for one isolated at 50°C accumulated mutations. Mutations were serially accumulated: Glu255Val, Asn43Tyr, Ala62Thr, Asn110Lys, and Ala112Val, respectively, at each step. The analyses of residual activity after heat treatment and the denaturation profile as monitored by circular dichroism showed that thermal stability was increased with accumulation of the mutations. The kinetic parameters of most mutant enzymes were similar to those of the wild type. However, some mutant enzymes showed a reverse correlation between stability and activity: the enzymes with a large increase in thermal stability showed lower activity. Although the wild-type enzyme is unstable in the absence of glycerol, the stabilizing effect of glycerol was not observed for all the mutant enzymes containing the Glu255Val substitution, which is assumed to be located at the hydrophobic interface between two subunits.