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Masayori Inouye

Publications and source records attributed to Masayori Inouye.

At least 37 records · Page 2Linked to original sources
PubMed2005

Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium.

In this chapter we describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples using Escherichia coli host vectors. The platform is centered on 6X-His affinity-tagged protein constructs, allowing for a similar purification procedure for most targets, and the implementation of high-throughput parallel methods. In most cases, these affinity-purified proteins are sufficiently homogeneous that a single subsequent gel filtration chromatography step is adequate to produce protein preparations that are greater than 98% pure. Using this platform, over 1000 different proteins have been cloned, expressed, and purified in tens of milligram quantities over the last 36-month period (see Summary Statistics for All Targets, ). Our experience using a hierarchical multiplex expression and purification strategy, also described in this chapter, has allowed us to achieve success in producing not only protein samples but also many three-dimensional structures. As of December 2004, the NESG Consortium has deposited over 145 new protein structures to the Protein Data Bank (PDB); about two-thirds of these protein samples were produced by the NESG Protein Production Facility described here. The methods described here have proven effective in producing quality samples of both eukaryotic and prokaryotic proteins. These improved robotic and?or parallel cloning, expression, protein production, and biophysical screening technologies will be of broad value to the structural biology, functional proteomics, and structural genomics communities.

Chromatography, Gel↗
PubMed2005

IRIP, a new ischemia/reperfusion-inducible protein that participates in the regulation of transporter activity.

We report the identification and characterization of a new ischemia/reperfusion-inducible protein (IRIP), which belongs to the SUA5/YrdC/YciO protein family. IRIP cDNA was isolated in a differential display analysis of an ischemia/reperfusion-treated kidney RNA sample. Mouse IRIP mRNA was expressed in all tissues tested, the highest level being in the testis, secretory, and endocrine organs. Besides ischemia/reperfusion, endotoxemia also activated the expression of IRIP in the liver, lung, and spleen. The transporter regulator RS1 was identified as an IRIP-interacting protein in yeast two-hybrid screening. The interaction between IRIP and RS1 was further confirmed in coimmunoprecipitation assays. A possible role of IRIP in regulating transporter activity was subsequently investigated. IRIP overexpression inhibited endogenous 1-methyl-4-phenylpyridinium (MPP+) uptake activity in HeLa cells. The activities of exogenous organic cation transporters (OCT2 and OCT3), organic anion transporter (OAT1), and monoamine transporters were also inhibited by IRIP. Conversely, inhibition of IRIP expression by small interfering RNA or antisense RNA increased MPP+ uptake. We measured transport kinetics of OCT2-mediated uptake and demonstrated that IRIP overexpression significantly decreased V(max) but did not affect K(m). On the basis of these results, we propose that IRIP regulates the activity of a variety of transporters under normal and pathological conditions.

Amino Acid Sequence↗
PubMed2004

Insights into the mRNA cleavage mechanism by MazF, an mRNA interferase.

MazF is an Escherichia coli toxin that is highly conserved among the prokaryotes and plays an important role in growth regulation. When MazF is induced, protein synthesis is effectively inhibited. However, the mechanism of MazF action has been controversial. Here we unequivocally demonstrate that MazF is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. We then demonstrate its enzymatic specificity using short RNA substrates. MazF cleaves RNA at the 5'-end of ACA sequences, yielding a 2',3'-cyclic phosphate at one side and a free 5'-OH group at the other. Using DNA-RNA chimeric substrates containing XACA, the 2'-OH group of residue X was found absolutely essential for MazF cleavage, whereas all the other residues may be deoxyriboses. Therefore, MazF exhibits exquisite site specificity and has utility as an RNA-restriction enzyme for RNA structural studies or as an mRNA interferase to regulate cell growth in prokaryotic and eukaryotic cells.

Base Sequence↗
PubMed2004

Complex formation between a putative 66-residue thumb domain of bacterial reverse transcriptase RT-Ec86 and the primer recognition RNA.

Reverse transcriptases (RT) are found in a minor population of Escherichia coli and are responsible for the synthesis of multicopy single-stranded DNA. These RTs specifically recognize RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branching G residue). Here, we purified the 66-residue, C-terminal fragment of RT-Ec86, RT from E. coli, which is responsible for the synthesis of multicopy single-stranded DNA-Ec86. This fragment, RT-Ec86-(255-320), was found to consist mainly of alpha-helical structures on the basis of its CD spectrum, which is consistent with the prediction of this region as the thumb domain from the structural alignment of RT-Ec86 with human immunodeficiency virus-1 RT. RT-Ec86-(255-320) was able to bind to a 28-base synthetic RNA consisting of the 5'-end single-stranded RNA containing the branching G residue and the recognition stem-loop structure in the RT-Ec86 primer-template RNA with a Kd value of 5 x 10(-8) M. By stepwise shortening of the 5'-end single-stranded region of the RNA, RT-Ec86-(255-320) was found still to be able to form a stable complex with only the stem-loop structure consisting of an 8-bp stem and a 3-base loop. In this stem-loop structure, the UUU loop was essential for the complex formation. RT-Ec73-(251-316) from another E. coli RT could not bind to the 28-base RNA for RT-Ec86 but could bind to its own stem-loop structure having a 3-base AGU loop. These results support the notion that the highly diverse C-terminal regions of bacterial RTs play an important role in recognizing their own specific primer-template RNA structure for the cDNA priming reaction.

Amino Acid Sequence↗
PubMed2004

The HAMP linker in histidine kinase dimeric receptors is critical for symmetric transmembrane signal transduction.

The HAMP linker, a common structural element between a sensor and a transmitter module in various sensor proteins, plays an essential role in signal transduction. Here, by in vivo complementation experiments with Tar-EnvZ hybrid receptor mutants in which the HAMP linker forms a heterodimer with Tar and EnvZ-type subunits, we found that mutations at one linker only affect the function of EnvZ in the same subunit. However, the same mutations affect the EnvZ function of both subunits when only a Tar or EnvZ-type HAMP linker is used. These results suggest that intersubunit interactions in the HAMP linker normally mediate signal transduction through both subunits in a sensor dimer, whereas the signal is asymmetrically transduced through the linker in a heterodimer. This is the first demonstration that two HAMP linkers in a sensor dimer are functionally coupled for normal signal transduction; however, this functional coupling can be reduced when the HAMP linkers lose their symmetric nature.

Bacterial Outer Membrane Proteins↗
PubMed2004

Cold-shock induced high-yield protein production in Escherichia coli.

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.

Amino Acid Sequence↗
PubMed2004

Cold shock response and adaptation at near-freezing temperature in microorganisms.

Microorganisms that naturally encounter sharp temperature shifts must develop strategies for responding and adapting to these shifts. Escherichia coli, which are adapted to living at both warm temperatures inside animals and cooler ambient temperatures, respond to low temperatures (10 degrees to 15 degrees C) by adjusting membrane lipid composition and increasing the production of proteins that act as "RNA chaperones" required for transcription and translation and proteins that facilitate ribosomal assembly. In contrast, yeast, which are adapted to cooler temperatures, show a relatively minor cold shock response after temperature shifts from 30 degrees to 10 degrees C but respond with a dramatic increase in the synthesis of trehalose and a heat shock protein when exposed to freezing or near-freezing temperatures. This emphasizes the fact that different groups of microorganisms exhibit distinct types of cold shock responses.

Adaptation, Physiological↗
PubMed2004

Interference of mRNA function by sequence-specific endoribonuclease PemK.

In Escherichia coli, programmed cell death is mediated through the system called "addiction module," which consists of a pair of genes encoding a stable toxin and a labile antitoxin. The pemI-pemK system is an addiction module present on plasmid R100. It helps to maintain the plasmid by post-segregational killing in E. coli population. Here we demonstrate that purified PemK, the toxin encoded by the pemI-pemK addiction module, inhibits protein synthesis in an E. coli cell-free system, whereas the addition of PemI, the antitoxin against PemK, resumes the protein synthesis. Further studies reveal that PemK is a sequence-specific endoribonuclease that cleaves mRNAs to inhibit protein synthesis, whereas PemI blocks the endoribonuclease activity of PemK. PemK cleaves only single-stranded RNA preferentially at the 5' or 3' side of the A residue in the "UAH" sequences (where H is C, A, or U). Upon induction, PemK cleaves cellular mRNAs to effectively block protein synthesis in E. coli. The pemK homologue genes have been identified on the genomes of a wide range of bacteria. We propose that PemK and its homologues form a novel endoribonuclease family that interferes with mRNA function by cleaving cellular mRNAs in a sequence-specific manner.

Base Sequence↗
PubMed2004

The mechanism of nucleic acid melting by a CspA family protein.

Cold-shock proteins of the CspA family help bacterial cells to acclimate to low temperatures. Some Csps bind single-stranded nucleic acids and destabilize nucleic acid secondary structures in vitro, and act as transcription antiterminators in vivo and in vitro. Nucleic acid melting by Escherichia coli CspE is critical for its ability to support low-temperature survival of the cell. Here, we explore the molecular mechanism of nucleic acid melting using CspE mutants harboring substitutions in surface-exposed residues critical for this function. Analysis of the mutants identifies two intermediates of the melting pathway and shows that CspE Phe17 and Phe30 act at the earliest stages of melting, while His32 acts later and is necessary for the propagation of melting. The results allow us to orient a CspE molecule relative to the melting substrate and to put forward a mechanistic model of nucleic acid melting by Csps.

Cold Temperature↗
PubMed2004

Genome-wide transcriptional analysis of the cold shock response in wild-type and cold-sensitive, quadruple-csp-deletion strains of Escherichia coli.

A DNA microarray-based global transcript profiling of Escherichia coli in response to cold shock showed that in addition to the known cold shock-inducible genes, new genes such as the flagellar operon, those encoding proteins involved in sugar transport and metabolism, and remarkably, genes encoding certain heat shock proteins are induced by cold shock. In the light of strong reduction in metabolic activity of the cell after temperature downshift, the induction of sugar metabolism machinery is unexpected. The deletion of four csps (cspA, cspB, cspG, and cspE) affected cold shock induction of mostly those genes that are transiently induced in the acclimation phase, emphasizing that CspA homologues are essential in the acclimation phase. Relevance of these findings with respect to the known RNA chaperone function of CspA homologues is discussed.

Bacterial Proteins↗
PubMed2003

The role of RbfA in 16S rRNA processing and cell growth at low temperature in Escherichia coli.

RbfA, a 30S ribosome-binding factor, is a multicopy suppressor of a cold-sensitive C23U mutation of the 16S rRNA and is required for efficient processing of the 16S rRNA. At 37 degrees C, DeltarbfA cells show accumulation of ribosomal subunits and 16S rRNA precursor with a significantly reduced polysome profile in comparison with wild-type cells. RbfA is also a cold-shock protein essential for Escherichia coli cells to adapt to low temperature. In this study, we examined its association with the ribosome and its role in 16S rRNA processing and ribosome profiles at low temperature. In wild-type cells, following cold shock at 15 degrees C, the amount of free RbfA remained largely stable, while that of its 30S subunit-associated form became several times greater than that at 37 degrees C and a larger fraction of total 30S subunits was detected to be RbfA-containing. In DeltarbfA cells, the pre-16S rRNA amount increased after cold shock with a concomitant reduction of the mature 16S rRNA amount and the formation of polysomes was further reduced. A closer examination revealed that 30S ribosomal subunits of DeltarbfA cells at low temperature contained primarily pre-16S rRNA and little mature 16S rRNA. Our results indicate that the cold sensitivity of DeltarbfA cells is directly related to their lack of translation initiation-capable 30S subunits containing mature 16S rRNA at low temperature. Importantly, when the C-terminal 25 residue sequence was deleted, the resulting RbfADelta25 lost the abilities to stably associate with the 30S subunit and to suppress the dominant-negative, cold-sensitive phenotype of the C23U mutation in 16S rRNA but was able to suppress the 16S rRNA processing defect and the cold-sensitive phenotype of the DeltarbfA cells, suggesting that RbfA may interact with the 30S ribosome at more than one site or function in more than one fashion in assisting the 16S rRNA maturation at low temperature.

Cell Division↗
PubMed2003

Characterization of the interactions within the mazEF addiction module of Escherichia coli.

In bacteria, programmed cell death is mediated through the unique genetic system called "addiction module," which consists of a pair of genes encoding a stable toxin and an unstable antitoxin. The mazEF system is known as an addiction module located on the Escherichia coli chromosome. MazF is a stable toxin, and MazE is a labile antitoxin interacting with MazF to form a complex. MazE and the MazE-MazF complex can bind to the mazEF promoter region to regulate the mazEF expression. Here we show that the binding of purified (His)6MazE to the mazEF promoter DNA was enhanced by MazF. The site-directed mutations at the conserved amino acid residues in MazE N-terminal region (K7A, R8A, S12A, and R16A) disrupted the DNA binding ability of both (His)6MazE and the MazE-MazF-(His)6 complex, suggesting that MazE binds to the mazEF promoter DNA through the N-terminal domain. The ratio of MazE to MazF(His)6 in the MazE-MazF(His)6 complex is about 1:2. Because both MazE and MazF-(His)6 exist as dimers by themselves, the MazE-MazF-(His)6 complex (76.9 kDa) is predicted to consist of one MazE dimer and two MazF(His)6 dimers. The interaction between MazE and MazF was also characterized with the yeast two-hybrid system. It was found that the region from residues 38 to 75 of MazE was required for its binding to MazF. Site-directed mutagenesis at this region revealed that Leu55 and Leu58 play an important role in the MazE-MazF complex formation but not in MazE binding to the mazEF promoter DNA. The present results demonstrate that MazE is composed of two domains, the N-terminal DNA-binding domain and the C-terminal domain interacting with MazF.

Amino Acid Sequence↗
PubMed2003

Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ).

The EnvZ/OmpR histidyl-aspartyl phosphorelay (HAP) system in Escherichia coli regulates the expression of ompF and ompC, the major outer membrane porin genes, in response to environmental osmolarity changes. Here, we report that dimers of EnvZc, the cytoplasmic domain of EnvZ, undergo spontaneous subunit exchange in solution. By introducing a cysteine substitution (S260C) in the dimerization domain of EnvZc, we were able to crosslink the two subunits in a dimer and trap the heterodimer formed between two different mutant EnvZc. By using a complementing system with two autophosphorylation-defective EnvZc mutants, one containing the H243V mutation at the autophosphorylation site and the other containing the G405A mutation in the ATP-binding domain, we demonstrated that an EnvZc(G405A) subunit can be phosphorylated by an EnvZc(H243V) subunit only when a heterodimer is formed. The rate of subunit exchange is concentration-dependent, with higher rates at higher concentrations of protein. The disulfide-crosslinked EnvZc(G405A) homodimer could not be phosphorylated by EnvZc(H243V), since the heterodimer formation between the two mutant proteins was blocked, indicating that autophosphorylation cannot occur by dimer-dimer interaction. By using MBP-deltaL-EnvZc(S260C) fusion protein (deltaL: the linker region, spanning residues 180-222, was deleted), it was found that in the disulfide-crosslinked MBP-deltaL-EnvZc(S260C)/deltaL-EnvZc(S260C/G405A) heterodimer, only the deltaL-EnvZc(S260C/G405A) subunit was phosphorylated but not the MBP-deltaL-EnvZc(S260C) subunit. Together, the present results provide biochemical evidence that EnvZ autophosphorylation occurs in trans and only within an EnvZ dimer.

Bacterial Outer Membrane Proteins↗
PubMed2003

Probing catalytically essential domain orientation in histidine kinase EnvZ by targeted disulfide crosslinking.

EnvZ, a dimeric transmembrane histidine kinase, belongs to the family of His-Asp phosphorelay signal transduction systems. The cytoplasmic kinase domain of EnvZ can be dissected into two independently functioning domains, A and B, whose NMR solution structures have been individually determined. Here, we examined the topological arrangement of these two domains in the EnvZ dimer, a structure that is key to understanding the mechanism underlying the autophosphorylation activity of the kinase. A series of cysteine substitution mutants were constructed to test the feasibility of chemical crosslinking between the two domains. These crosslinking data demonstrate that helix I of domain A of one subunit in the EnvZc dimer is in close proximity to domain B of the other subunit in the same dimer, while helix II of domain A of one subunit interacts with domain B of the same subunit in the EnvZc dimer. This is the first demonstration of the topological arrangement between the central dimerization domain containing the active center His residues (domain A) and the ATP-binding catalysis assisting domain (domain B) in a class I histidine kinase.

Bacterial Outer Membrane Proteins↗
PubMed2003

Analysis of the role of the EnvZ linker region in signal transduction using a chimeric Tar/EnvZ receptor protein, Tez1.

Tez1 is a chimeric protein in which the periplasmic and transmembrane domains of Tar, a chemosensor, are fused to the cytoplasmic catalytic domain of EnvZ, an osmosensing histidine kinase, through the EnvZ linker. Unlike Taz1 (a similar hybrid with the Tar linker), Tez1 could not respond to Tar ligand, aspartate, whereas single Ala insertion at the transmembrane/linker junction, as seen in Tez1A1, restored the aspartate-regulatable phenotype. Analysis of the Ala insertion site requirement and the nature of the insertion residue on the phenotype of Tez1 indicated that a junction region between the transmembrane domain and the predicted helix I in the linker is critical to signal transduction. Random mutagenesis revealed that P185Q mutation in the Tez1 linker restored the aspartate-regulatable phenotype. Substitution mutations at Pro-185 further demonstrated that specific residues are required at this site for an aspartate response. None of the hybrid receptors constructed with different Tar/EnvZ fusion sites in the linker could respond to aspartate, suggesting that specific interactions between the two predicted helices in the linker are important for the linker function. In addition, a mutation (F220D) known to cause an OmpCc phenotype in EnvZ resulted in similar OmpCc phenotypes in both Tez1A1 and Tez1, indicating the importance of the predicted helix II in signal propagation. Together, we propose that the N-terminal junction region modulates the alignment between the two helices in the linker upon signal input. In turn helix II propagates the resultant conformational signal into the downstream catalytic domain of EnvZ to regulate its bifunctional enzymatic activities.

Alanine↗
PubMed2003

Thermotoga maritima MazG protein has both nucleoside triphosphate pyrophosphohydrolase and pyrophosphatase activities.

MazG proteins form a widely conserved family among bacteria, but their cellular function is still unknown. Here we report that Thermotoga maritima MazG protein (Tm-MazG), the product of the TM0913 gene, has both nucleoside triphosphate pyrophosphohydrolase (NTPase) and pyrophosphatase activities. Tm-MazG catalyzes the hydrolysis of all eight canonical ribo- and deoxyribonucleoside triphosphates to their corresponding nucleoside monophosphates and PPi and subsequently hydrolyzes the resultant PPi to Pi. The NTPase activity with deoxyribonucleoside triphosphates as substrate is higher than corresponding ribonucleoside triphosphates. dGTP is the best substrate among the deoxyribonucleoside triphosphates, and GTP is the best among the ribonucleoside triphosphates. Both NTPase and pyrophosphatase activities were enhanced at higher temperatures and blocked by the alpha,beta-methyleneadenosine triphosphate, which cannot be hydrolyzed by Tm-MazG. Furthermore, PPi is an inhibitor for the Tm-MazG NTPase activity. Significant decreases in the NTPase activity and concomitant increases in the pyrophosphatase activity were observed when mutations were introduced at the highly conserved amino acid residues in Tm-MazG N-terminal region (E41Q/E42Q, E45Q, E61Q, R97A/R98A, and K118A). These results demonstrated that Tm-MazG has dual enzymatic functions, NTPase and pyrophosphatase, and that these two enzymatic activities are coordinated.

Adenosine Triphosphate↗
PubMed2003

Solution NMR structure of ribosome-binding factor A (RbfA), a cold-shock adaptation protein from Escherichia coli.

Ribosome-binding factor A (RbfA) from Escherichia coli is a cold-shock adaptation protein. It is essential for efficient processing of 16S rRNA and is suspected to interact with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is a member of a large family of small proteins found in most bacterial organisms, making it an important target for structural proteomics. Here, we describe the three-dimensional structure of RbfADelta25, a 108 residue construct with 25 residues removed from the carboxyl terminus of full-length RbfA, determined in solution at pH 5.0 by heteronuclear NMR methods. The structure determination was carried out using largely automated methods for determining resonance assignments, interpreting nuclear Overhauser effect (NOE) spectroscopy (NOESY) spectra, and structure generation. RbfADelta25 has an alpha+beta fold containing three helices and three beta-strands, alpha1-beta1-beta2-alpha2-alpha3-beta3. The structure has type-II KH-domain fold topology, related to conserved KH sequence family proteins whose betaalphaalphabeta subunits are characterized by a helix-turn-helix motif with sequence signature GxxG at the turn. In RbfA, this betaalphaalphabeta subunit is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, including a strongly conserved Ala residue at position 75 forming an interhelical kink. The electrostatic field distribution about RbfADelta25 is bipolar; one side of the molecule is strongly negative and the opposite face has a strong positive electrostatic field. A "dynamic hot spot" of RbfADelta25 has been identified in the vicinity of a beta-bulge at strongly conserved residue Ser39 by 15N R(1), R(2) relaxation rate and heteronuclear 15N-1H NOE measurements. Analyses of these distributions of electrostatic field and internal dynamics, together with evolutionary implications of fold and sequence conservation, suggest that RbfA is indeed a nucleic acid-binding protein, and identify a potential RNA-binding site in or around the conserved polypeptide segment Ser76-Asp100 corresponding to the alpha3-loop-beta3 helix-loop-strand structure. While the structure of RbfADelta25 is most similar to that of the KH domain of the E.coli Era GTPase, its electrostatic field distribution is most similar to the KH1 domain of the NusA protein from Thermotoga maritima, another cold-shock associated RNA-binding protein. Both RbfA and NusA are regulated in the same E.coli operon. Structural and functional similarities between RbfA, NusA, and other bacterial type II KH domains suggest previously unsuspected evolutionary relationships between these cold-shock associated proteins.

Adaptation, Physiological↗