Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and non-competitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, co-immunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affect the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction, and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms.



<bold>Importance</bold>



Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization, and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry, and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.

DOI： 10.1128/jvi.00938-21

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.bbrc.2021.08.070

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：National Academy of Sciences  

Chloride ion–pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl− into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation–diffusion process upon light-triggered retinal isomerization.

DOI： 10.1073/pnas.2020486118

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer  

Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.

DOI： 10.1007/978-981-15-8763-4_7

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Language：English   Publishing type：Research paper (scientific journal)  

SeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac[Formula: see text](2-3)Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc) and its precursor, asialo-GM1 (Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the [Formula: see text]-trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

DOI： 10.1038/s41598-020-78926-7

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Nucleus accumbens-associated protein 1 (NAC1) is a nuclear protein that harbors an amino-terminal BTB domain and a carboxyl-terminal BEN domain. NAC1 appears to play significant and diverse functions in cancer and stem cell biology. Here we demonstrated that the BEN domain of NAC1 is a sequence-specific DNA-binding domain. We selected the palindromic 6 bp motif ACATGT as a target sequence by using a PCR-assisted random oligonucleotide selection approach. The interaction between NAC1 and target DNA was characterized by gel shift assays, pull-down assays, isothermal titration calorimetry (ITC), chromatin-immunoprecipitation assays, and NMR chemical shifts perturbation (CSP). The solution NMR structure revealed that the BEN domain of human NAC-1 is composed of five conserved α helices and two short β sheets, with an additional hitherto unknown N-terminal α helix. In particular, ITC clarified that there are two sequential events in the titration of the BEN domain of NAC1 into the target DNA. The ITC results were further supported by CSP data and structure analyses. Furthermore, live cell photobleaching analyses revealed that the BEN domain of NAC1 alone was unable to interact with chromatin/other proteins in cells.

DOI： 10.3390/biomedicines8120608

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>
The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.

DOI： 10.1038/s41467-020-18559-6

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Other Link： http://www.nature.com/articles/s41467-020-18559-6

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jmb.2020.07.016

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Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

Hemoglobin is one of the best-characterized proteins with respect to structure and function, but the internal ligand diffusion pathways remain obscure and controversial. Here we captured the CO migration processes in the tense (T), relaxed (R), and second relaxed (R2) quaternary structures of human hemoglobin by crystallography using a high-repetition pulsed laser technique at cryogenic temperatures. We found that in each quaternary structure, the photodissociated CO molecules migrate along distinct pathways in the α and β subunits by hopping between the internal cavities with correlated side chain motions of large nonpolar residues, such as α14Trp(A12), α105Leu(G12), β15Trp(A12), and β71Phe(E15). We also observe electron density evidence for the distal histidine [α58/β63His(E7)] swing-out motion regardless of the quaternary structure, although less evident in α subunits than in β subunits, suggesting that some CO molecules have escaped directly through the E7 gate. Remarkably, in T-state Fe(II)-Ni(II) hybrid hemoglobins in which either the α or β subunits contain Ni(II) heme that cannot bind CO, the photodissociated CO molecules not only dock at the cavities in the original Fe(II) subunit, but also escape from the protein matrix and enter the cavities in the adjacent Ni(II) subunit even at 95 K, demonstrating the high gas permeability and porosity of the hemoglobin molecule. Our results provide a comprehensive picture of ligand movements in hemoglobin and highlight the relevance of cavities, nonpolar residues, and distal histidines in facilitating the ligand migration.

DOI： 10.1073/pnas.1913663117

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Other Link： https://syndication.highwire.org/content/doi/10.1073/pnas.1913663117

Language：Japanese   Publisher：(公社)日本薬学会  

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Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

A newly identified microbial rhodopsin, NM-R3, from the marine flavobacterium<italic>Nonlabens marinus</italic>, was recently shown to drive chloride ion uptake, extending our understanding of the diversity of mechanisms for biological energy conversion. To clarify the mechanism underlying its function, we characterized the crystal structures of NM-R3 in both the dark state and early intermediate photoexcited states produced by laser pulses of different intensities and temperatures. The displacement of chloride ions at five different locations in the model reflected the detailed anion-conduction pathway, and the activity-related key residues—Cys¹⁰⁵, Ser⁶⁰, Gln²²⁴, and Phe⁹⁰—were identified by mutation assays and spectroscopy. Comparisons with other proteins, including a closely related outward sodium ion pump, revealed key motifs and provided structural insights into light-driven ion transport across membranes by the NQ subfamily of rhodopsins. Unexpectedly, the response of the retinal in NM-R3 to photostimulation appears to be substantially different from that seen in bacteriorhodopsin.

DOI： 10.1126/sciadv.aay2042

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Language：English   Publishing type：Research paper (scientific journal)  

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4β (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.

DOI： 10.1074/jbc.AC119.010366

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DOI： 10.1016/j.bbrc.2019.01.142

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Sodium taurocholate cotransporting polypeptide (NTCP) is a host cell receptor required for hepatitis B virus (HBV) entry. However, the susceptibility of NTCP-expressing cells to HBV is diverse depending on the culture condition. Stimulation with epidermal growth factor (EGF) was found to potentiate cell susceptibility to HBV infection. Here, we show that EGF receptor (EGFR) plays a critical role in HBV virion internalization. In EGFR-knockdown cells, HBV or its preS1-specific fluorescence peptide attached to the cell surface, but its internalization was attenuated. PreS1 internalization and HBV infection could be rescued by complementation with functional EGFR. Interestingly, the HBV/preS1-NTCP complex at the cell surface was internalized concomitant with the endocytotic relocalization of EGFR. Molecular interaction between NTCP and EGFR was documented by immunoprecipitation assay. Upon dissociation from functional EGFR, NTCP no longer functioned to support viral infection, as demonstrated by either (i) the introduction of NTCP point mutation that disrupted its interaction with EGFR, (ii) the detrimental effect of decoy peptide interrupting the NTCP-EGFR interaction, or (iii) the pharmacological inactivation of EGFR. Together, these data support the crucial role of EGFR in mediating HBV-NTCP internalization into susceptible cells. EGFR thus provides a yet unidentified missing link from the cell-surface HBV-NTCP attachment to the viral invasion beyond the host cell membrane.

DOI： 10.1073/pnas.1811064116

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DOI： 10.3389/fmicb.2018.03257

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Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.

DOI： 10.1016/j.bbrc.2018.04.187

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Lippincott Williams and Wilkins  

Objective - Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1β (interleukin 1β) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. Approach and Results - The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1β release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1β release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1β release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1β release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1β release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1β-deficient mice. Conclusions - These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.

DOI： 10.1161/ATVBAHA.117.310581

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Language：English   Publishing type：Research paper (scientific journal)  

Current anti-hepatitis B virus (HBV) agents including interferons and nucleos(t)ide analogs efficiently suppress HBV infection. However, as it is difficult to eliminate HBV from chronically infected liver, alternative anti-HBV agents targeting a new molecule are urgently needed. In this study, we applied a chemical array to high throughput screening of small molecules that interacted with sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for HBV. From approximately 30,000 compounds, we identified 74 candidates for NTCP interactants, and five out of these were shown to inhibit HBV infection in cell culture. One of such compound, NPD8716, a coumarin derivative, interacted with NTCP and inhibited HBV infection without causing cytotoxicity. Consistent with its NTCP interaction capacity, this compound was shown to block viral attachment to host hepatocytes. NPD8716 also prevented the infection with hepatitis D virus, but not hepatitis C virus, in agreement with NPD8716 specifically inhibiting NTCP-mediated infection. Analysis of derivative compounds showed that the anti-HBV activity of compounds was apparently correlated with the affinity to NTCP and the capacity to impair NTCP-mediated bile acid uptake. These results are the first to show that the chemical array technology represents a powerful platform to identify novel viral entry inhibitors.

DOI： 10.1038/s41598-018-20987-w

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Although X-ray crystallography is the most commonly used technique for studying the molecular structure of proteins, it is not generally able to monitor the dynamic changes or global domain motions that often underlie allostery. These motions often prevent crystal growth or reduce crystal order. We have recently discovered a crystal form of human hemoglobin that contains three protein molecules allowed to express a full range of quaternary structures, whereas maintaining strong X-ray diffraction. Here we use this crystal form to investigate the effects of two allosteric effectors, phosphate and bezafibrate, by tracking the structures and functions of the three hemoglobin molecules following the addition of each effector. The X-ray analysis shows that the addition of either phosphate or bezafibrate not only induces conformational changes in a direction from a relaxed-state to a tense-state, but also within relaxed-state populations. The microspectrophotometric O-2 equilibrium measurements on the crystals demonstrate that the binding of each effector energetically stabilizes the lowest affinity conformer more strongly than the intermediate affinity one, thereby reducing the O-2 affinity of tense-state populations, and that the addition of bezafibrate causes an approximate to 5-fold decrease in the O-2 affinity of relaxed-state populations. These results show that the allosteric pathway of hemoglobin involves shifts of populations rather than a unidirectional conversion of one quaternary structure to another, and that minor conformers of hemoglobin may have a disproportionate effect on the overall O-2 affinity.

DOI： 10.1074/jbc.M117.781146

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light-and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

DOI： 10.1073/pnas.1704391114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Survival of Clonorchis sinensis, a cause of human clonorchiasis, requires tegument proteins, which are localized to the tegumental outer surface membrane. These proteins play an important role in a host response and parasite survival. Thus, these proteins are interesting molecular targets for vaccine and drug development. Here, we have determined two crystal structures of the calmodulin like domain (amino acid [aa] positions 1-81) and dynein light chain (DLC)-like domain (aa 83-177) of a 20.8-kDa tegumental-allergen-like protein from Clonorchis sinensis (CsTAL3). The calmodulin like domain has two Ca2+-binding sites (named CB1 and CB2), but Ca2+ binds to only one site, CB1. The DLC-like domain has a dimeric conformation; the interface is formed mainly by hydrogen bonds between the main chain atoms. In addition, we have determined full-length structure of CsTAL3 in solution and showed the conformational change of CsTAL3 induced by Ca2+ ion binding using small-angle X-ray scattering analysis and molecular dynamics simulations. The Ca2+-bound form has a more extended conformation than the Ca2+-free from does. These structural and biochemical analyses will advance the understanding of the biology of this liver fluke and may contribute to our understanding of the molecular mechanism of calcium-responsive and tegumental-allergen-like proteins.

DOI： 10.1038/s41598-017-02044-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

Nucleosomes are dynamic entities that are repositioned along DNA by chromatin remodeling processes. A nucleosome repositioned by the switch-sucrose nonfermentable (SWI/SNF) remodeler collides with a neighbor and forms the intermediate" overlapping dinucleosome." Here, we report the crystal structure of the overlapping dinucleosome, in which two nucleosomes are associated, at 3.14-angstrom resolution. In the overlapping dinucleosome structure, the unusual "hexasome" nucleosome, composed of the histone hexamer lacking one H2A-H2B dimer from the conventional histone octamer, contacts the canonical "octasome" nucleosome, and they intimately associate. Consequently, about 250 base pairs of DNA are left-handedly wrapped in three turns, without a linker DNA segment between the hexasome and octasome moieties. The overlapping dinucleosome structure may provide important information to understand how nucleosome repositioning occurs during the chromatin remodeling process.

DOI： 10.1126/science.aak9867

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DOI： 10.1016/j.jhep.2016.11.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (K-d = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research.

DOI： 10.1038/srep43480

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

MytiLec is a lectin, isolated from bivalves, with cytotoxic activity against cancer cell lines that express globotriaosyl ceramide, Gal alpha(1,4)Gal beta(1,4) Glc alpha 1-Cer, on the cell surface. Functional analysis shows that the protein binds to the disaccharide melibiose, Gal alpha(1,6)Glc, and the trisaccharide globotriose, Gal alpha(1,4)Gal beta(1,4)Glc. Recombinant MytiLec expressed in bacteria showed the same haemagglutinating and cytotoxic activity against Burkitt's lymphoma (Raji) cells as the native form. The crystal structure has been determined to atomic resolution, in the presence and absence of ligands, showing the protein to be a member of the beta-trefoil family, but with a mode of ligand binding unique to a small group of related trefoil lectins. Each of the three pseudo-equivalent binding sites within the monomer shows ligand binding, and the protein forms a tight dimer in solution. An engineered monomer mutant lost all cytotoxic activity against Raji cells, but retained some haemagglutination activity, showing that the quaternary structure of the protein is important for its cellular effects.

DOI： 10.1038/srep28344

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Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α2β2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm3) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

DOI： 10.1063/1.4953453

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 angstrom across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

DOI： 10.1073/pnas.1517520113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Anti-hepatitis B virus (HBV) drugs are currently limited to nucleos(t)ide analogs (NAs) and interferons. A challenge of drug development is the identification of small molecules that suppress HBV infection from new chemical sources. Here, from a fungus-derived secondary metabolite library, we identify a structurally novel tricyclic polyketide, named vanitaracin A, which specifically inhibits HBV infection. Vanitaracin A inhibited the viral entry process with a submicromolar 50% inhibitory concentration (IC50) (IC50 = 0.61 +/- 0.23 mu M), without evident cytotoxicity (50% cytotoxic concentration of &gt;256 mu M; selectivity index value of &gt;419) in primary human hepatocytes. Vanitaracin A did not affect the HBV replication process. This compound was found to directly interact with the HBV entry receptor sodium taurocholate cotransporting polypeptide (NTCP) and impaired its bile acid transport activity. Consistent with this NTCP targeting, antiviral activity of vanitaracin A was observed with hepatitis D virus (HDV) but not hepatitis C virus. Importantly, vanitaracin A inhibited infection by all HBV genotypes tested (genotypes A to D) and clinically relevant NA-resistant HBV isolate. Thus, we identified a fungal metabolite, vanitaracin A, which was a potent, well-tolerated, and broadly active inhibitor of HBV and HDV entry. This compound, or its related analogs, could be part of an antiviral strategy for preventing reinfection with HBV, including clinically relevant nucleos(t) ide analog-resistant virus.
IMPORTANCE
For achieving better treatment and prevention of hepatitis B virus (HBV) infection, anti-HBV agents targeting a new molecule are in great demand. Although sodium taurocholate cotransporting polypeptide (NTCP) has recently been reported to be an essential host factor for HBV entry, there is a limited number of reports that identify new compounds targeting NTCP and inhibiting HBV entry. Here, from an uncharacterized chemical library, we isolated a structurally new compound, named vanitaracin A, which inhibited the process of entry of HBV and hepatitis D virus (HDV). This compound was suggested to directly interact with NTCP and inhibit its transporter activity. Importantly, vanitaracin A inhibited the entry of all HBV genotypes examined and of a clinically relevant nucleos(t) ide analog-resistant HBV isolate.

DOI： 10.1128/JVI.01855-15

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3 ' splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the beta sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein.

DOI： 10.1101/gad.267104.115

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Rap1B is a small GTPase involved in the regulation of numerous cellular processes including synaptic plasticity, one of the bases of memory. Like other members of the Ras family, the active GTP-bound form of Rap1B can bind to a large number of effector proteins and so transmit signals to downstream components of the signaling pathways. The structure of Rap1B bound only to a nucleotide has yet to be solved, but might help reveal an inactive conformation that can be stabilized by a small molecule drug. Unlike other Ras family proteins such as H-Ras and Rap2A, Rap1B crystallizes in an intermediate state when bound to a non-hydrolyzable GTP analog. Comparison with H-Ras and Rap2A reveals conservative mutations relative to Rap1B, distant from the bound nucleotide, which control how readily the protein may adopt the fully activated form in the presence of GTP. High resolution crystallographic structures of mutant proteins show how these changes may influence the hydrogen bonding patterns of the key switch residues. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

DOI： 10.1016/j.bbrc.2015.04.103

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The X-ray crystal structure of chitin oligosaccharide deacetylase from Vibrio parahaemolyticus (Vp-COD) was determined at an 1.35 angstrom resolution. The amino acid sequence and structure of Vp-COD show that the enzyme comprises one polysaccharide deacetylase domain (PDD) and two carbohydrate-binding domains (CBDs). On the basis of a chitin-binding assay with Vp-COD and its CBDs-deleted mutant, it was confirmed that CBDs can adhere to chitin. The catalytic activity of the CBDs-deleted mutant was only mildly depressed compared with that of Vp-COD, indicating that CBDs are unlikely to affect the configuration of the active center residues in active site of PDD. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2014.11.039

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The modular structure of many protein families, such as beta-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms. Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical beta-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2-10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.

DOI： 10.1073/pnas.1412768111

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Blood clotting is a vitally important process that must be carefully regulated to prevent blood loss on one hand and thrombosis on the other. Severe injury and hemophilia may be treated with pro-coagulants, whereas risk of obstructive clotting or embolism may be reduced with anti-coagulants. Anti-coagulants are an extremely important class of drug, one of the most widely used types of medication, but there remains a pressing need for novel treatments, however, as present drugs such as warfarin have significant drawbacks. Nature provides a number of examples of anti-coagulant proteins produced by blood-sucking animals, which may provide templates for the development of new small molecules with similar physiological effects. We have, therefore, studied an Anopheles anti-platelet protein from a malaria vector mosquito and report its crystal structure in complex with an antibody. Overall the protein is extremely sensitive to proteolysis, but the crystal structure reveals a stable domain built from two helices and a turn, which corresponds to the functional region. The antibody raised against Anopheles anti-platelet protein prevents it from binding collagen. Our work, therefore, opens new avenues to the development of both novel small molecule anti-clotting agents and anti-malarials.

DOI： 10.1074/jbc.M114.564526

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Allostery in many oligomeric proteins has been postulated to occur via a ligand-binding-driven conformational transition from the tense (T) to relaxed (R) state, largely on the basis of the knowledge of the structure and function of 2 1 hemoglobin, the most thoroughly studied of all allosteric proteins. However, a growing body of evidence suggests that hemoglobin possesses a variety of intermediates between the two end states. As such intermediate forms coexist with the end states in dynamic equilibrium and cannot be individually characterized by conventional techniques, very little is known about their properties and functions. Here we present complete structural and functional snapshots of nine equilibrium conformers of human hemoglobin in the halfliganded and fully liganded states by using a novel combination of X-ray diffraction analysis and microspectrophotometric 02 equilibrium measurements on three isomorphous crystals, each capturing three distinct equilibrium conformers. Notably, the conformational set of this crystal form varies according to shifts in the allosteric equilibrium, reflecting the differences in hemoglobin ligation state and crystallization solution conditions. We find that nine snapshot structures cover the complete conformational space of hemoglobin, ranging from T to R2 (the second relaxed quaternary structure) through R, with various relaxed intermediate forms between R and R2. Moreover, we find a previously unidentified intermediate conformer, between T and R, with an intermediate O2 affinity, sought by many research groups over a period of decades. These findings reveal a comprehensive picture of the equilibrium conformers and transition pathway for human hemoglobin.

DOI： 10.1021/ja500380e

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S254_6

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The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (ß 1, ß 2 and ß 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the ß5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a ß5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.

DOI： 10.1371/journal.pone.0060649

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Other Link： http://orcid.org/0000-0002-7249-6418

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Bacterial biofilm formation is an extremely widespread phenomenon involving the secretion of a protective exopolysaccharide matrix which helps the bacteria to attach to surfaces and to overcome a variety of stresses in different environments. This matrix may also include proteins, lipids, DNA and metal ions. Its composition depends on the bacterial species and growth conditions, but one of the most widely found components is polymeric beta-1,6-N-acetyl-d-glucosamine (PGA). Several studies have suggested that PGA is an essential component of biofilm and it is produced by numerous bacteria, including Escherichia coli, Staphylococcus epidermis, Yersinia pestis, Bordetella spp. and Actinobacillus spp. In E. coli, PGA production and export are dependent on four genes that form a single operon, pgaABCD, which appears to have been transferred between various species. Biofilms themselves are recognized as environments in which such horizontal gene transfer may occur. The pga operon of E. coli, which is even found in innocuous laboratory strains, is highly homologous to that from the plague bacterium Yersinia pestis, and biofilm is believed to play an important role in the transmission of Yersinia. The crystal structure of the N-terminal domain of PgaB, which has deacetylase activity, is described and compared with models of other deacetylases.

DOI： 10.1107/S0907444912042059

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The haemoglobin (Hb) of the extinct woolly mammoth has been recreated using recombinant genes expressed in Escherichia coli. The globin gene sequences were previously determined using DNA recovered from frozen cadavers. Although highly similar to the Hb of existing elephants, the woolly mammoth protein shows rather different responses to chloride ions and temperature. In particular, the heat of oxygenation is found to be much lower in mammoth Hb, which appears to be an adaptation to the harsh high-latitude climates of the Pleistocene Ice Ages and has been linked to heightened sensitivity of the mammoth protein to protons, chloride ions and organic phosphates relative to that of Asian elephants. To elucidate the structural basis for the altered homotropic and heterotropic effects, the crystal structures of mammoth Hb have been determined in the deoxy, carbonmonoxy and aquomet forms. These models, which are the first structures of Hb from an extinct species, show many features reminiscent of human Hb, but underline how the delicate control of oxygen affinity relies on much more than simple overall quaternary-structure changes.

DOI： 10.1107/S0907444912029459

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Staphylococcus aureus is a widespread Gram-positive opportunistic pathogen, and a methicillin-resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin-binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the beta-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin-binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of "dimerization domain", the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but Mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2012.07.012

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Human noroviruses are genetically and antigenically highly divergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-Å resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at ∼10-Å resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised ∼15 Å off the shell domain and rotated ∼40° relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in interactions with other regions of the capsid and is buried in the virus particle. Despite the occluded nature of the recognized epitope in the VLP structure, enzyme-linked immunosorbent assay (ELISA) binding suggested that the 5B18 antibody was able to capture intact VLPs. Together, the results provide evidence that the norovirus particle is capable of extreme conformational flexibility, which may allow for antibody recognition of conserved surfaces that would otherwise be buried on intact particles.

DOI： 10.1128/JVI.06868-11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (beta 6 Glu -&gt; Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 angstrom resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K-4) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2-3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.

DOI： 10.1074/jbc.M111.266056

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：7359  

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

DOI： 10.1038/nature10258

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Language：Japanese   Publisher：The Crystallographic Society of Japan  

Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. Influenza virus reproduces rapidly, mutates frequently, and occasionally crosses species barriers. The recent emergence of swine-origin influenza H1N1 and avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here, we describe two crystal structures of complexes made by fragments of PA and PB1, and PB1 and PB2. These novel interfaces are surprisingly small, yet they play a crucial role in regulating the 250 kDa polymerase complex, and are completely conserved among swine, avian and human influenza viruses. Given their importance to viral replication and strict conservation, the PA/PB1 and PB1/PB2 interfaces appear to be promising targets for novel anti-influenza drugs of use against all strains of influenza A virus. It is hoped that the structures presented here will assist the search for such compounds.

DOI： 10.5940/jcrsj.52.271

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Other Link： https://jlc.jst.go.jp/DN/JALC/00362367834?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

The autotransporter Tsh (temperature-sensitive haemagglutinin) secreted by avian pathogenic Escherichia coli was reported in 1994 and the almost identical Hbp (haemoglobin protease) was discovered some years later in isolates from patients suffering from peritoneal abscesses. However, the function of the protein remains uncertain. The crystal structure of Hbp shows that the protein carries a serine protease domain (domain 1) and a small domain of 75 residues called domain 2 which is inserted into the long beta-helix characteristic of autotransporter passenger proteins. In this paper, domain 1 is shown to bind calcium, although metal ions binding to this site do not seem to regulate protease activity. Tsh has been reported to bind red cells and components of the extracellular matrix, but it is demonstrated that these properties are not a consequence of the presence of domain 2.

DOI： 10.1107/S0907444910036966

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Publisher：Pt 11  

DOI： 10.1107/S0907444910025448

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Many virulence factors secreted by pathogenic Gram-negative bacteria are found to be members of the autotransporter protein family. These proteins share a common mechanism by which they exit the periplasm, involving the formation of a 12-stranded beta-barrel domain in the outer membrane. The role of this barrel in the secretion of the N-terminal passenger domain is controversial, and no model currently explains satisfactorily the entire body of experimental data. After secretion, some autotransporter barrels autoproteolytically cleave away the passenger, and one crystal structure is known for a barrel of this type in the postcleavage state. Hbp is an autotransporter of the self-cleaving type, which cuts the polypeptide between two absolutely conserved asparagine residues buried within the barrel lumen. Mutation of the first asparagine residue to isosteric aspartic acid prevents proteolysis. Here we present the crystal structure of a truncated Hbp mutant carrying the C-terminal residues of the passenger domain attached to the barrel. This model mimics the state of the protein immediately prior to separation of the passenger and barrel domains, and shows the role of residues in the so-called "linker" between the passenger and beta domains. This high-resolution membrane protein crystal structure also reveals the sites of many water molecules within the barrel. The cleavage mechanism shows similarities to those of inteins and some viral proteins, but with a novel means of promoting nucleophilic attack. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2010.06.068

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Other Link： http://orcid.org/0000-0003-0909-0789

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

The Streptomyces butyrolactone autoregulator receptor protein (BarA) is a DNA-binding protein that regulates the biosynthesis of the antibiotic virginiamycin. In this study, BarA from S. virginiae was overexpressed in Escherichia coli, purified and crystallized. Crystals of purified protein have been grown that diffracted to beyond 3.0 angstrom resolution at 100 K using synchrotron radiation. The protein crystals belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 128.0, c = 286.2 angstrom. With four molecules per asymmetric unit, the crystal volume per unit protein mass (V(M)) was 3.2 angstrom(3) Da(-1) and the solvent content was 62%.

DOI： 10.1107/S1744309110009930

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Other Link： http://orcid.org/0000-0003-0909-0789

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DOI： 10.1016/j.jmb.2010.03.016

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. Influenza virus reproduces rapidly, mutates frequently, and occasionally crosses species barriers. The recent emergence of swine-origin influenza H1N1 and avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. I describe two crystal structures of complexes made by fragments of PA and PB1, and PB1 and PB2. These novel interfaces are surprisingly small, yet they play a crucial role in regulating the 250 kDa polymerase complex, and are completely conserved among swine, avian and human influenza viruses. Given their importance to viral replication and strict conservation, the PA/PB1 and PB1/PB2 interfaces appear to be promising targets for novel anti-influenza drugs of use against all strains of influenza A virus. It is hoped that the structures presented here will assist the search for such compounds.<br>

DOI： 10.2142/biophys.50.074

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Other Link： https://jlc.jst.go.jp/DN/JALC/00348858424?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel beta-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo, form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by beta-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2009.11.055

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Microtubule plus-end tracking proteins (+TIPs) control microtubule dynamics in fundamental processes such as cell cycle, intracellular transport, and cell motility, but how +TIPs are regulated during mitosis remains largely unclear. Here we show that the endogenous end-binding protein family EB3 is stable during mitosis, facilitates cell cycle progression at prometaphase, and then is down-regulated during the transition to G(1) phase. The ubiquitin-protein isopeptide ligase SIAH-1 facilitates EB3 polyubiquitination and subsequent proteasome-mediated degradation, whereas SIAH-1 knockdown increases EB3 stability and steady-state levels. Two mitotic kinases, Aurora-A and Aurora-B, phosphorylate endogenous EB3 at Ser-176, and the phosphorylation triggers disruption of the EB3-SIAH-1 complex, resulting in EB3 stabilization during mitosis. Our results provide new insight into a regulatory mechanism of +TIPs in cell cycle transition.

DOI： 10.1074/jbc.M109.000273

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Influenza virus RNA-dependent RNA polymerase is a multi-functional heterotrimer, which uses a 'cap-snatching' mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap-bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap-binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA-binding PB1 subunit to the cap-binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo-enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses. The EMBO Journal (2009) 28, 1803-1811. doi:10.1038/emboj.2009.138; Published online 21 May 2009

DOI： 10.1038/emboj.2009.138

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Escherichia coli PBP4 is the archetypal class C, low molecular mass penicillin binding protein (LMM-PBP) and possesses both DD-carboxypeptidase and DD-endopeptidase activity. In contrast to other 14 classes of PBP, class C LMM-PBPs show high DD-carboxypeptidase activity and rapidly hydrolyze synthetic fragments of peptidoglycan. The recently solved X-ray crystal structures of three class C LMM-PBPs (E. coli PBP4, Bacillus subtilis PBP4a, and Actinomadura R39 DD-peptidase) have identified several residues that form a pocket in the active site unique to this class of PBP. The X-ray cocrystal structure of the Actinomadura R39 DD-peptidase with a cephalosporin bearing a peptidoglycan-mimetic side chain showed that residues of this pocket interact with the third position meso-2,6-diaminopimelic acid residue of the peptidoglycan stem peptide. Equivalent residues of E. coli PBP4 (Asp 155, Phe160, Arg361, and Gln422) were mutated, and the effect on both DD-carboxypeptidase and DD-endopeptidase activities was determined. Using N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-meso-2,6-diaminopimelyl-D-alanyl-D-alanine as substrate, mutation of Asp155, Phe160, Arg361, and Gln422 to alanine reduced k(cat)/K(m) by 12.7-, 1.9-, 24.5-,, and 13.8-fold, respectively. None of the k(cat) values deviated significantly from wild-type PBP4. PBP4 DD-endopeptidase activity was also affected, with substitution of Asp 155, Arg361, and Gln422 reducing specific activity by 22%, 56%, and 40%, respectively. This provides the first direct demonstration of the importance of residues forming a subsite to accommodate meso-2,6-diaminopimelic acid in both the DD-carboxypeptidase and DD-endopeptidase activities of a class C LMM-PBP.

DOI： 10.1021/bi801993x

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DOI： 10.1021/ac802354j

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Language：Japanese   Publisher：The Crystallographic Society of Japan  

DOI： 10.5940/jcrsj.51.104

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DOI： 10.1073/pnas.0801032106

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Proteins harbor a number of cavities of relatively small volume. Although these packing defects are associated with the thermodynamic instability of the proteins, the cavities also play specific roles in controlling protein functions, e.g., ligand migration and binding. This issue has been extensively studied in a well-known protein, myoglobin (Mb). Mb reversibly binds gas ligands at the heme site buried in the protein matrix and possesses several internal cavities in which ligand molecules can reside. It is still an open question as to how a ligand finds its migration pathways between the internal cavities. Here, we report on the dynamic and sequential structural deformation of internal cavities during the ligand migration process in Mb. Our method, the continuous illumination of native carbonmonoxy Mb crystals with pulsed laser at cryogenic temperatures, has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino acid residues around the cavity, which results in the expansion of the cavity with a breathing motion. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand migration channel arising by induced fit, which is further supported by computational geometry analysis by the Delaunay tessellation method. This result suggests a crucial role of the breathing motion of internal cavities as a general mechanism of ligand migration in a protein matrix.

DOI： 10.1073/pnas.0807774106

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Language：Japanese   Publisher：The Crystallographic Society of Japan  

Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently, and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2, and PA are required for both transcription and replication. PB1 carries the polymerise active site, PB2 includes the capped-RNA recognition domain, and PA is involved in assembly of the functional complex, but so far very little structural information has been reported for any of them. We describe the crystal structure of a large fragment of one subunit (PA) of influenza A RNA polymerise bound to a fragment of another subunit (PB1) . The C terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the N-terminal residues of PB1 can fit by forming a 3<SUB>10</SUB> helix.

DOI： 10.5940/jcrsj.50.329

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DOI： 10.2210/pdb3ecz/pdb

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Other Link： http://orcid.org/0000-0002-7119-2017

DOI： 10.2210/pdb2zso/pdb

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Other Link： http://orcid.org/0000-0002-7119-2017

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S5_2

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To characterize a diheme cytochrome <I>c</I><SUB>4</SUB> of unknown functional of the <I>Vibrio</I> genus for the first time, the <I>Vibrio parahaemolyticus</I> cytochrome <I>c</I><SUB>4</SUB> was overexpressed in <I>Escherichia coli</I> periplasm using the endogenous signal sequence. The physicochemical properties of the purified recombinant protein, <I>viz.</I>, molecular mass, UV/Vis, and CD spectra, and the redox potentials of the N- and C-terminal domain hemes were determined.

DOI： 10.1271/bbb.80380

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Other Link： https://jlc.jst.go.jp/DN/JALC/00319864669?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments(1). Here we demonstrate the importance to viral replication of a subunit interface in the viral RNA polymerase, thereby providing a new set of potential drug binding sites entirely independent of surface antigen type. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2 and PA, are required for both transcription and replication(2-4). PB1 carries the polymerase active site, PB2 includes the capped- RNA recognition domain, and PA is involved in assembly of the functional complex(5-7), but so far very little structural information has been reported for any of them(8-11). We describe the crystal structure of a large fragment of one subunit ( PA) of influenza A RNA polymerase bound to a fragment of another subunit ( PB1). The carboxy- terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the amino- terminal residues of PB1 can fit by forming a 3(10) helix.

DOI： 10.1038/nature07225

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Language：English  

DOI： 10.1074/jbc.M708684200

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Other Link： http://orcid.org/0000-0001-7412-3722

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DOI： 10.1016/j.jmb.2008.01.020

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DOI： 10.1110/ps.073059308

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DOI： 10.1021/ja0749441

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The denaturation of recombinant horse L-chain apoferritin (rLF), which is composed of 24 L-chain subunits, in acidic solution was studied. Using two rLF mutants, lacking four (Fer4) or eight (Fer8) N-terminal amino acid residues, the effect of N-terminal residues on the protein's stability was investigated. Of the two mutants and wild-type rLF, the tertiary and secondary structures of Fer8 were found to be most sensitive to an acidic environment. The Fer8 protein dissociated easily into subunit dimers at or below pH 2.0. Comparing the crystal structures of the mutant proteins, deletion of the N-terminal residues was found to result in fewer inter- and intra-subunit hydrogen bonds. The loss of these bonds is assumed to be responsible for lower endurance against acidic denaturation in N-terminus-deleted mutants. These results indicated that the inter- and intra-subunit hydrogen bonds of N-terminal residues affect the denaturation, especially oligomer formation of apoferritin subunits and will be of use in designing ferritin-based nanodevices.

DOI： 10.1093/jb/mvm187

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DOI： 10.1128/JB.00713-07

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Publisher：Pt 9  

DOI： 10.1107/S1744309107033854

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

We have discovered distinct, characteristic differences in the thermodynamic signatures of tryptophan binding by trp RNA-binding attenuation protein (TRAP) from two different bacterial species. The TRAP 11mer ring binds 11 molecules of tryptophan at symmetry-related sites. Tryptophan binding to Bacillus stearothermophilus TRAP is not cooperative, but isothermal titration calorimetry shows that filling the first tryptophan binding sites of Bacillus subtilis TRAP has a marked effect on the thermodynamics of subsequent ligand binding. We have identified a single, conservative amino acid replacement (Ile to Leu) in B. subtilis TRAP that abolishes this effect, and suggest the initial ligand binding causes a change throughout the wildtype protein ring. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2007.05.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

HpcG catalyses the hydration of a carbon-carbon double bond without the aid of any cofactor other than a simple divalent metal ion such as Mg2+. Since the substrate has a nearby carbonyl group, it is believed that it first isomerises to form a pair of conjugated double bonds in the enol tautomer before Michael addition of water. Previous chemical studies of the reaction, of the mechanism. The substrate itself is unstable, preventing co-crystallisation or soaking of crystals, but oxalate is a strong competitive inhibitor. We have solved the crystal structure of the protein in the apo form, and with magnesium and oxalate bound. Modelling substrate into the active site suggests the attacking water molecule is not part of the metal coordination shell, in contrast to a previous proposal. Our model suggests that geometrically strained cis isomer intermediates do not lie on the reaction pathway, and that separate groups are involved in the isomerisation and hydration steps. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2007.05.006

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DOI： 10.1107/S0907444906048712

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S27_3

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Language：English   Publishing type：Research paper (scientific journal)  

The gene encoding 2-oxo-hept-3-ene-1,7-dioic acid (OHED) hydratase (HpcG) was cloned into the high-expression plasmid pET26b and overexpressed in Escherichia coli BL21(DE3). The enzyme was purified in three steps to greater than 95% purity prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K in a number of screening conditions. Crystals measuring up to 1.5 mm in their longest dimension were grown from solutions containing polyethylene glycol 20 000. The crystals belonged to space group P41212 or P432 12, with unit-cell parameters a = 136, b = 136, c = 192 Å. A complete data set was collected to 2.1 Å from a single cryocooled crystal at 100 K using synchrotron radiation. © International Union of Crystallography, 2006.

DOI： 10.1107/S1744309106035901

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Publisher：3  

DOI： 10.1016/j.jmb.2006.05.036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The protein TRAP (trp RNA binding attenuation protein) forms a highly thermostable ring-shaped 11-mer. By linking in tandem two, three, or four DNA sequences encoding TRAP monomers, we have engineered new rings that consist of 12 TRAP subunits and bind 12 ligand molecules. The hydrogen bonding pattern and buried surface area within and between subunits are essentially identical between the 11-mer and 12-mer crystal structures. Why do the artificial proteins choose to make single 12-mer rings? The 12-mer rings are highly sterically strained by their peptide linkers and far from thermostable. That proteins choose to adopt a strained conformation of few subunits rather than an unstrained one with 11 subunits demonstrates the importance of entropic factors in controlling protein-protein interactions in general.

DOI： 10.1016/j.str.2006.03.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The crystal structure of penicillin binding protein 4 (PBP4) from Escherichia coli, which has both DD-endopeptidase and DD-carboxypeptidase activity, is presented. PBP4 is one of 12 penicillin binding proteins in E. coli involved in the synthesis and maintenance of the cell wall. The model contains a penicillin binding domain similar to known structures, but includes a large insertion which folds into domains with unique folds. The structures of the protein covalently attached to five different antibiotics presented here show the active site residues are unmoved compared to the apoprotein, but nearby surface loops and helices are displaced in some cases. The altered geometry of conserved active site residues compared with those of other PBPs suggests a possible cause for the slow deacylation rate of PBP4.

DOI： 10.1021/bi051533t

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The acquisition of iron is essential for the survival of pathogenic bacteria, which have consequently evolved a wide variety of uptake systems to extract iron and heme from host proteins such as hemoglobin. Hemoglobin protease (Hbp) was discovered as a factor involved in the symbiosis of pathogenic Escherichia coli and Bacteroides fragilis, which cause intra-abdominal abscesses. Released from E. coli, this serine protease autotransporter degrades hemoglobin and delivers heme to both bacterial species. The crystal structure of the complete passenger domain of Hbp (110 kDa) is presented, which is the first structure from this class of serine proteases and the largest parallel beta-helical structure yet solved.

DOI： 10.1074/jbc.M412885200

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The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel β-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins. Copyright © 2005 The Protein Society.

DOI： 10.1110/ps.041183305

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S36_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S285_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

The first successful multi-wavelength anomalous diffraction ( MAD) experiments using ultra-high-energy X-rays ( similar to 35 keV) were performed for iodine and xenon derivatives of hen egg-white lysozyme crystals. The beamline BL41XU of SPring-8 enabled the collection of high-quality MAD data, which led to the calculation of anomalous or dispersive difference Patterson maps that determined the positions of iodine and xenon atoms. The electron density maps obtained by the density modification method for both cases proved to be of sufficient quality for building molecular models. I-MAD and Xe-MAD phasing are now available at SPring-8, and the utilization of ultra-high-energy X-rays will make a significant contribution to the solution of the phase problem in protein crystallography.

DOI： 10.1107/S0021889804023076

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

DOI： 10.1002/prot.20282

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Publisher：27  

DOI： 10.1021/bi049932w

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The crystal structure of hemoglobin has been known for several decades, yet various features of the molecule remain unexplained or controversial. Several animal hemoglobins have properties that cannot be readily explained in terms of their amino acid sequence and known atomic models of hemoglobin. Among these, fish hemoglobins are well known for their widely varying interactions with heterotropic effector molecules and pH sensitivity. Some fish hemoglobins are almost completely insensitive to pH (within physiological limits), whereas others show extremely low oxygen affinity under acid conditions, a phenomenon called the Root effect. X-ray crystal structures of Root effect hemoglobins have not, to date, provided convincing explanations of this effect. Sequence alignments have signally failed to pinpoint the residues involved, and site-directed mutagenesis has not yielded a human hemoglobin variant with this property. We have solved the crystal structure of tuna hemoglobin in the deoxy form at low and moderate pH and in the presence of carbon monoxide at high pH. A comparison of these models shows clear evidence for novel mechanisms of pH-dependent control of ligand affinity.

DOI： 10.1074/jbc.M401740200

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DOI： 10.1002/prot.20122

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The most frequent modification of RNA, the conversion of uridine bases to pseudouridines, is found in all living organisms and often in highly conserved locations in ribosomal and transfer RNA. RluC and RluD are homologous enzymes which each convert three specific uridine bases in Escherichia coli ribosomal 23S RNA to pseudouridine: bases 955, 2504, and 2580 in the case of RluC and 1911, 1915, and 1917 in the case of RluD. Both have an N-terminal S4 RNA binding domain. While the loss of RluC has little phenotypic effect, loss of RluD results in a much reduced growth rate. We have determined the crystal structures of the catalytic domain of RluC, and full-length RluD. The S4 domain of RluD appears to be highly flexible or unfolded and is completely invisible in the electron density map. Despite the conserved topology shared by the two proteins, the surface shape and charge distribution are very different. The models suggest significant differences in substrate binding by different pseudouridine synthases.

DOI： 10.1021/bi036079c

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S111_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S111_1

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S134_3

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S20_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Bacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel. Nickel homeostasis systems include the dedicated nickel uptake system nik found in Escherichia coli, a member of the ABC family of transporters, that involves a periplasmic nickel-binding protein, NikA. This is the initial nickel receptor and mediator of the chemotactic response away from nickel. We have solved the crystal structure of NikA protein in the presence and absence of nickel, showing that it behaves as a "classical" periplasmic binding protein. In contrast to other binding proteins, however, the ligand remains accessible to the solvent and is not completely enclosed. No direct bonds are formed between the metal cation and the protein. The nickel binding site is apolar, quite unlike any previously characterized protein nickel binding site. Despite relatively weak binding, NikA is specific for nickel. Using isothermal titration calorimetry, the dissociation constant for nickel was found to be similar to 10 muM and that for cobalt was approximately 20 times higher.

DOI： 10.1074/jbc.M307941200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (&gt;95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (&gt;95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O-2 and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.

DOI： 10.1021/bi035410p

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DOI： 10.1074/jbc.M310388200

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DOI： 10.1074/jbc.M304339200

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Publisher：8  

DOI： 10.1110/gad.03104003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

TT1466 is a hypothetical protein from the extremely thermophilic bacterium Thermus thermophilus HB8 and is highly conserved in bacteria and archaea. The selenomethionyl protein was synthesized by a cell-free system and the crystal structure was determined at 2.0 Angstrom by MAD phasing. A native crystal was used for structure refinement to 1.7 Angstrom. The structure is highly homologous to that of the CoA-binding domain of the succinyl-CoA synthetase from Escherichia coli, despite the protein having only 14% sequence identity to this domain. An isothermal titration calorimetry experiment was performed to investigate whether TT1466 binds CoA and revealed high-affinity CoA binding of TT1466.

DOI： 10.1107/S0907444903011028

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Publisher：12  

DOI： 10.1073/pnas.1230629100

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Cytochrome P450 isolated from Bacillus subtilis (P450(BSbeta); molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the alpha- and beta-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450(BSbeta) in the substrate-bound form, determined at a resolution of 2.1 Angstrom P450(BSbeta) exhibits a typical P450 fold. The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg(242) guanidium group. These interactions are responsible for the site specificity of the hydroxylation site in which the a- and U-positions of the fatty acid come into close proximity to the heme iron sixth site. The fatty acid carboxylate group interacts with Arg(242) in the same fashion as has been reported for the active site of chloroperoxidase, His(105)Glu(183), which is an acid-base catalyst in the peroxidation reactions. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450(BSbeta) is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.

DOI： 10.1074/jbc.M211575200

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DOI： 10.1107/S0907444902017705

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Publisher：41  

DOI： 10.1074/jbc.M205461200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Crystal structures of a thermostable cytochrome P450 (CYP119) and a site-directed mutant, (Phe24Leu), from the acidothermophilic archaea Sulfolobus solfataricus were determined at 1.5-2.0 Angstrom resolution. We identify important crystallographic waters in the ferric heme pocket, observe protein conformational changes upon inhibitor binding, and detect a unique distribution of surface charge not found in other P450s. An analysis of factors contributing to thermostability of CYP119 of these high resolution structures shows an apparent increase in clustering of aromatic residues and optimum stacking. The contribution of aromatic stacking was investigated further with the mutant crystal structure and differential scanning calorimetry. (C) 2002 Elsevier Science Inc. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Hagfish are extremely primitive jawless fish of disputed ancestry. Although generally classed with lampreys as cyclostomes ("round mouths"), it is clear that they diverged from them several hundred million years ago. The crystal structures of the deoxy and CO forms of hemoglobin from a hagfish (Eptatretus burgeri) have been solved at 1.6 and 2.1 Angstrom, respectively. The deoxy crystal contains one dimer and two monomers in a unit cell, with the dimer being similar to that found in lamprey deoxy-Hb, but with a larger interface and different relative orientation of the partner chains. Ile(E11) and Gln(E7) obstruct ligand binding in the deoxy form and make room for ligands in the CO form, but no interaction path between the two hemes could be identified. The BGH core structure, which forms the a, 13, interface of all vertebrate alpha(2)beta(2) tetrameric Hbs, is conserved in hagfish and lamprey Hbs. It was shown previously that human and cartilaginous fish Hbs have independently evolved stereochemical mechanisms other than the movement of the proximal histidine to regulate ligand binding at the hemes. Our results therefore suggest that the formation of the alpha(2)beta(2) tetramer using the BGH core and the mechanism of quaternary structure change evolved between the branching points of hagfish and lampreys from other vertebrates.

DOI： 10.1074/jbc.M111492200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

Cytochrome P450 isolated from Bacillus subtilis (P450BSbeta; MW 48 kDa) catalyzes the hydroxylation of long-chain fatty acids at the alpha and beta positions using H2O2 as an oxidant. Crystals of the substrate-free form of P450BSbeta belonging to the trigonal space group P3(2)21 or P3(1)21 were obtained by the sitting-drop vapour-diffusion method using a precipitate solution consisting of 10%(w/v) PEG 4000 and 50 mM MES pH 6.8. Another crystal form, belonging to the rhombohedral space group R3 or R32, was obtained from precipitate solution consisting of 10% PEG 4000, 0.15 mM magnesium acetate and 50 mM MES pH 6.5 in the presence of 2 mM myristic acid (substrate). Using synchrotron radiation, both P450BSbeta crystals diffracted to 2.5 Angstrom resolution. Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the haem iron.

DOI： 10.1107/S0907444902001762

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

Crystal structures of the nitric oxide reductase cytochrome P450nor (P450nor) in the ferric resting and the ferrous carbonmonoxy (CO) states have been determined at 1.00 and 1.05 Angstrom resolution, respectively. P450nor consists of 403 aminoacid residues (46 kDa) and is one of the largest proteins refined to this resolution so far. The final models have conventional R factors of 10.2% (ferric resting) and 11.7% (ferrous CO), with mean coordinate errors of 0.028 (ferric resting) and 0.030 Angstrom (ferrous CO) as calculated from inversion of the full positional least-squares matrix. Owing to the atomic resolution, novel features are found in the refined structures. Firstly, two orientations of the haem are observed both in the ferric resting and the ferrous CO states. Secondly, a disordered water molecule bound to the haem iron is found in the ferric resting state. In addition, the accurate structures at atomic resolution enabled the examination of general stereochemical parameters that are commonly used in refinement cycles of protein structures.

DOI： 10.1107/S0907444901017383

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S128_4

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ELSEVIER SCIENCE BV  

Nitric oxide reductase (NOR) isolated from Fusarium oxysporum catalyzes the reduction reaction of nitric oxide (NO) to nitrous oxide (N2O). In this reaction, the transient reaction intermediate (t=similar to20 s) is produced by the reduction of ferric-NO complex with NADH. To elucidate the electronic structure of the intermediate, we measured its resonance Raman spectrum using the continuous-flow method. From these spectral features, we have concluded that the electronic structure of the intermediate in the NO reduction reaction by fungal NOR is Fe2+NO-H+. Further, we have found that the Ser286/Asp393 hydrogen-bond network is the proton pathway to the active site on the basis of crystallographic and mutagenesis studies. From these results, we discuss the molecular mechanism of NO reduction reaction catalyzed by fungal NOR. (C) 2002 Elsevier Science B.V. All rights reserved.

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Using UV-Vis, resonance Raman, and EPR spectroscopy we have studied the properties of the oxygenated ferrous cytochrome P450 from Sulfolobus Solfataricus, (CYP119). The recently determined crystal structure of CYP119 is compared with other available structures of P450s, and detailed structural and spectroscopic analyses are reported. With several structural similarities to CYP102, such as in-plane iron position and a shorter iron-proximal ligand bond, CYP119 shows low-spin conformation preference in the ferric form and partially in the ferrous form at low temperatures. These structural features can explain the fast autoxidation of the oxyferrous complex of CYP119. Finally, we report the first UV-Vis and EPR spectra of the cryoradiolytically reduced oxygenated intermediate of CYP119. The primary reduced intermediate, a hydroperoxo-ferric complex of CYP119, undergoes a 'peroxide shunt' pathway during gradual annealing at 170-195 K and returns to the low-spin ferric form. © 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0162-0134(01)00328-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MUNKSGAARD INT PUBL LTD  

The crystallization and preliminary crystallographic study of phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus is reported. Phycocyanin is composed of alpha- and beta -subunits consisting of 162 and 172 amino-acid residues, respectively. These associate to form an alpha beta heterodimer, which further associates to give a ring-shaped trimer (alpha beta)(3). Two trimers bind head-to-head to form a hexamer (alpha beta)(6). Phycocyanin crystals have been obtained by the sitting-drop vapour-diffusion method with a precipitant solution containing 30%(w/v) PEG 4000 and 100 mM MES pH 7.5-8.0. Using synchrotron radiation, the crystals diffract to 2.0 Angstrom resolution. They belong to the trigonal space group R32, with unit-cell parameters a = b = 186.75 (3), c = 59.75 (4) Angstrom, alpha = beta = 90, gamma = 120 degrees. Assuming that the crystallographic triad is identical to the threefold axis of the hexamer and with three (alpha beta)(6) molecules in a unit cell, the calculated molar volume (V-M) is 2.64 Angstrom (3) Da(-1). This value corresponds to a solvent content of approximately 53%, with one alpha beta heterodimer occupying the asymmetric unit.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A SPring-8 bending magnet beamline, the RIKEN structural biology beamline II (BL44B2), is dedicated to monochromatic/Laue macromolecular crystallography and X-ray absorption fine structure studies. The design and the performance of the beamline are presented. (C) 2001 Elsevier Science B.V. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD  

Although many descriptions of adaptive molecular evolution of vertebrate hemoglobins (Hb) can be found in physiological text books, they are based mainly on changes of the primary structure and place more emphasis on conservation than alterations at the functional site. Sequence analysis alone, however, does not reveal much about the evolution of new functions in proteins It was found recently that there are many functionally important structural differences between human and a ray (Dasyatis akajei) I FD even where sequence is conserved between the two. We have solved the structures of the deoxy and CO forms of a second cartilaginous fish (a shark, Mustelus griseus) Hb, and compared it with structures of human Hb, two bony fish Hbs and the ray Hb in order to understand more about how vertebrate Hbs have functionally evolved by the selection of random amino acid substitutions. The sequence identity of cartilaginous fish Hb and human Hb is a little less than 40 %, with many functionally important amino acid replacements. Wider substitutions than usually considered as neutral have been accepted in the course of molecular evolution of Hb. As with the ray Hb, the shark Hb shows functionally important structural differences from human Hb that involve amino acid substitutions and shifts of preserved amino acid residues induced by substitutions in other parts of the molecule. Most importantly, beta E11Val in deoxy human Hb, which overlaps the ligand binding site and is considered to play a key role in controlling the oxygen affinity, moves away about 1 A in both the shark and ray Hbs. Thus adaptive molecular evolution is feasible as a result of both functionally significant mutations and deviations of preserved amino acid residues induced by other amino acid substitutions. (C) 2001 Academic Press.

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S45_2

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S112_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

The structure-function relationship in cytochrome P450cam monooxygenase was studied by employing its active site mutant Thr252Ile, X-ray crystallographic analyses of the ferric d-camphor-bound form of the mutant revealed that the mutation caused a structural change in the active site giving an enlarged oxygen-binding pocket that did not contain any hydrophilic group such as the OH group of Thr and H2O. The enzyme showed a low monooxygenase activity of ca, 1/10 of the activity of the wild-type enzyme. Kinetic analyses of each catalytic step revealed that the rate of proton-coupled reduction of the oxygenated intermediate of the enzyme, a ternary complex of dioxygen and d-camphor with the ferrous enzyme, decreased to about 1/30 of that of the wild-type enzyme, while the rates of other catalytic steps including the reduction of the ferric d-camphor-bound form by reduced putidaredoxin did not change significantly. These results indicated that a hydrophilic group(s) such as water and/or hydroxyl group in the active site is prerecquisite to a proton supply for the reduction of the oxygenated intermediate, thereby giving support for the operation of a proton transfer network composed of Thr252, Asp251, and two other amino acids and water proposed by previous investigators.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MUNKSGAARD INT PUBL LTD  

CYP119 is a cytochrome P450 with a molecular weight of 43 kDa which has been isolated from the thermophilic archaeon Sulfolobus solfataricus. This enzyme is extremely stable to high temperature and high pressure. The first crystallization and preliminary crystallographic study of CYP119 is reported here. Crystals of CYP119 were obtained by the sitting-drop vapour-diffusion method using a precipitant solution containing 20%(w/v) PEG 4000 and 0.2 M sodium thiocyanate at pH 6.4. Using synchrotron radiation, the CYP119 crystal diffracted to 1.84 Angstrom resolution. It belongs to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 86.17 (0.07), c = 221.11 (0.04) Angstrom, in which the numbers in parentheses describe the standard deviations. Assuming two molecules of the CYP119 per asymmetric unit, the calculated molar volume (V-m) is 2.38 Angstrom(3) Da(-1). Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the haem irons. The complete crystallographically defined structure is currently in progress using MIR (multiple isomorphous replacement) and MAD (multiwavelength anomalous diffraction) techniques.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER-VERLAG TOKYO  

Nitric oxide reductase cytochrome P-450(nor) (P-450(nor)) isolated from Fusarium oxysporum is the heme enzyme catalyzing NO reduction in the fungal denitrification process. The three-dimensional structures of this P-450(nor) in the ferric resting and the ferrous-CO states were determined by X-ray diffraction and refined at 2.0 Angstrom to an R-factor of 19.7% and 19.9%, respectively. Although the overall structure of P-450(nor) is basically similar to those of monooxygenase cytochrome P-450s, the pocket in the heme distal side is widely opened, implicating the binding of NADH in this site. On binding of CO to the heme iron, the water molecules located in the heme distal pocket are rearranged in their positions, resulting in formation of the hydrogen bound network from the water molecule adjacent to the iron ligant to the protein surface of the distal pocket through the hydroxyl group of Ser-286 and the carboxyl group of Asp-393. Thus, this hydrogen-bonding network possibly provides a pathway of proton delivery in the NO reduction reaction.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Cytochrome P450nor (P450nor) is a heme-containing nitric oxide reductase from the denitrifying fungus, Fusarium oxysporum. This enzyme catalyzes the reduction of NO to N2O, In the present study, we report results from preliminary crystallographic and electron paramagnetic resonance (EPR) analysis of a single crystal of P450nor, The crystal was grown in 100 mM MES buffer at pH 5.6 using PEG 4000 as a precipitant, It belongs to the orthorhombic system with cell dimensions of a=54.99 Angstrom, b=82.66 Angstrom, c=87.21 Angstrom, and the space group is P2(1)2(1)2(1). The crystal diffracts synchrotron radiation at higher than 2.0 Angstrom resolution, and therefore it is suitable for X-ray crystal structure analysis at atomic resolution, Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the heme iron, The structure solution is currently underway bg means of MIR and MAD techniques, EPR analysis determined the orientation of the heme within the P450nor crystal. (C) 1997 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(97)00795-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Two isozymes of nitric oxide reductase (Nor) from the denitrifying fungus Cylindrocarpon tonkinense (c.Norl and c.Nor2) are the heme-enzyme cytochrome P-450's (Usuda et al. (1995) Appl. Environ. Microbiol. 61, 883-889). However, they catalyze the NO reduction to N2O, but not the monooxygenation reaction using O-2. We kinetically and spectrophotometrically studied the reactions of the two Nor's with NO and electron donor, NAD(P)H, using flash-photolysis and stopped-flow rapid scan methods, The enzyme in the Fe3+ state can bind NO to yield the Fe3+NO complex. When the resultant Fe3+ NO complex reacted with the electron donor, it was converted to the Fe3+ enzyme via a transient formation of the characteristic intermediate (I). The spectroscopic results were essentially the same as those of the Nor from another denitrifying fungus Fusarium oxysporum (f.Nor), which we previously reported (Shire et al. (1995) J. Biol. Chem. 270, 1617-1623), suggesting that these fungal Nor's catalyze the NO reduction by the same mechanism. Most probably, the Fe3+NO complex of the Nor is reduced with two-electrons directly transferred from NAD(P)H to yield the intermediate I, and then the I reacts with another NO to generate N2O and the Fe3+ enzyme. However, the kinetic measurements showed that the reaction rate constant of each step was variable depending on the combination of the Nor and the electron donor; i.e., c.Nor1 + NADH, c.Nor2 + NADPH, c.Nor2 + NADH and f.Nor + NADH. In particular, the rate constant for the electron transfer step from the electron donor to the Fe3+ NO enzyme is dramatically different among these systems. On the other hand, we also measured paramagnetically shifted H-NMR spectra of c.Nor2 and f.Nor in the ferrous (reduced) state, where the iron-bound Cys beta-CH2 signal was observed at the same position (approximate to 270 ppm) for c.Nor2 and f.Nor, indicating that the Cys thiolate (S-) coordinates to the heme iron in the same fashion in the Nor's. However, the heme peripheral proton signals were subtly but significantly different in their positions between the two enzymes. On the basis of these kinetic and spectroscopic data, we suggested that the Fe-S- binding character is not essential for the NO reduction reactivity, but that the subtle difference in interaction of their hemes with the surroundings is possibly responsible for the difference in the Nor reactivity, especially in the electron transfer step from NAD(P)H to the Fe3+ NO moiety.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD  

The structures of deoxy (alpha((Mg(II)))(2) beta((Fe(II)))(2)) and CO-Liganded (alpha((Mg(II)))(2)((Fe(II)-CO))(2)) forms of human hemoglobin were determined by X-ray crystallography to a resolution of 1.7 Angstrom and 1.9 Angstrom, respectively. The deoxy hybrid has virtually the same structure as that of the native deoxy HbA. Both the deoxy and CO-liganded hybrids assumed a T quaternary structure characteristic of native deoxy HbA. No significant structural difference was found between the a subunits of the CO-liganded hybrid and deoxy HbA, while in the beta subunit, significant tertiary structural changes were confined to the heme pocket. Baldwin showed by a comparison of COHbA and deoxy HbA that there is a 1.5 Angstrom shift of the beta subunit heme into its pocket; This shift was much reduced in alpha((Mg(II)))(2) beta((Fe(II)-CO))(2). On the other hand, when the two structures are compared with superposition of hemes, the nearest neighbors of CO (Fe, E11 Val and E7 His) have shifted nearly to the same positions as those in COHbA. Thus the tertiary structure of the beta subunit of alpha((Mg(II)))(2) beta((Fe(II)-CO))(2) is such that the CO molecule and the neighboring atoms assume nearly the same conformation as those of COHbA, while the block shift of these groups is impeded with respect to the structural invariant portions of the molecule. (C) 1996 Academic Press Limited.

DOI： 10.1006/jmbi.1996.0059

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Other Link： http://orcid.org/0000-0002-1700-7861

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

一般に,海洋細菌であるVibrio属細菌は,キチナーゼを分泌してN,N&#039;-diacetylchitobiose[(GlcNAc)_2]を生産することが知られている。しかし,一部の菌はさらにキチンオリゴ糖脱アセチル化酵素(COD)を分泌し,菌体外最終産物としてβ-N-acetyl-D-glucosaminyl-(1,4)-D-glucosamine(GlcNAc-GlcN)を生産する。CODは,Carbohydrate esterase family 4に分類され(GlcNAc)_2に特異性の高い脱アセチル化酵素として2010年にEC 3.5.1.105が付された酵素である。CODの酵素化学的性質については,数種のVibrio属細菌由来の酵素で報告されている。最近,我々はVibrio parahaemolyticus由来CODが1つの活性ドメイン(PDD)と2つのキチン結合ドメイン(CBD)から成ることを立体構造から確認した。また,各ドメインがそれぞれ独立して機能することも解明した。さらにGlcNAc-GlcNは,COD生産性Vibrio属細菌に特異的なキチナーゼ生産誘導因子および遊走性因子であることも明らかにした。これらのことから,CODは一部のVibrio属細菌にとってはキチン利用のためのシグナル分子生産に必須な酵素であることがわかった。

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Language：Japanese   Publisher：日本応用糖質科学会  

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Language：Japanese   Publisher：日本応用糖質科学会  

CiNii Books

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Language：Japanese   Publisher：日本応用糖質科学会  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：INT UNION CRYSTALLOGRAPHY  

DOI： 10.1107/S0108767311089677

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：INT UNION CRYSTALLOGRAPHY  

DOI： 10.1107/S0108767311089641

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：SPRINGER  

Autotransporter proteins are virulence factors associated with a wide variety of diseases caused by pathogenic Gram-negative bacteria, and they play a variety of roles in pathogenesis including disabling host defences and mediating colonization. Pertactin, a key component of the whooping cough vaccine, is an autotransporter protein. A large sub-family of the autotransporters carries a trypsin-like protease domain, but these enzymes have different substrates and functions. The unique export process which defines the autotransporter family involves the polypeptide chain C-terminus forming a barrel structure in the bacterial outer membrane, but the role of this barrel in secreting of the N-terminal &apos;passenger&apos; domain remains very unclear. There are now four published crystal structures of passenger proteins or fragments of them. We have compared these models to catalogue common features and to help predict the structures and functions of other autotransporter proteins such as SepA, which is involved in the pathogenicity of Shigella.

DOI： 10.1007/s00109-010-0600-y

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：化学同人  

CiNii Books

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Language：English   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

We determined for the first time the crystal structure of diatom cytochrome <I>c</I><SUB>6</SUB> from <I>Phaeodactylum tricornutum</I> at 1.5 Å resolution. The overall structure of the protein was classified as a class I <I>c</I>-type cytochrome. The physicochemical properties of the protein were examined by denaturation with guanidine hydrochloride and urea, and compared with those of other algal cytochrome <I>c</I><SUB>6</SUB>.

DOI： 10.1271/bbb.80472

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Other Link： https://jlc.jst.go.jp/DN/JALC/00324722886?from=CiNii

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：INT UNION CRYSTALLOGRAPHY  

DOI： 10.1107/S0108767308088545

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Language：English   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

Although detailed crystal structures of haemoglobin (Hb) provide a clear understanding of the basic allosteric mechanism of the protein, and how this in turn controls oxygen affinity, recent experiments with artificial effector molecules have shown a far greater control of oxygen binding than with natural heterotropic effectors. Contrary to the established text-book view, these non-physiological compounds are able to reduce oxygen affinity very strongly without switching the protein to the T (tense) state. In an earlier paper we showed that bezafibrate (BZF) binds to a surface pocket on the a. subunits of R state Hb, strongly reducing the oxygen affinity of this protein conformation. Here we report the crystallisation of Hb with L35, a related compound, and show that this binds to the central cavity of both R and T state Hb. The mechanism by which L35 reduces oxygen affinity is discussed, in relation to spectroscopic studies of effector binding. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2005.12.018

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DOI： 10.1016/j.febslet.2006.05.067

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Crystal structures of the ferric and ferrous heme complexes of HmuO, a 24-kDa heme oxygenase of Corynebacterium diphtheriae, have been refined to 1.4 and 1.5 Angstrom resolution, respectively. The HmuO structures show that the heme group is closely sandwiched between the proximal and distal helices. The imidazole group of His-20 is the proximal heme ligand, which closely eclipses the beta- and delta-meso axis of the porphyrin ring. A long range hydrogen bonding network is present, connecting the iron-bound water ligand to the solvent water molecule. This enables proton transfer from the solvent to the catalytic site, where the oxygen activation occurs. In comparison to the ferric complex, the proximal and distal helices move closer to the heme plane in the ferrous complex. Together with the kinked distal helix, this movement leaves only the alpha-meso carbon atom accessible to the iron-bound dioxygen. The heme pocket architecture is responsible for stabilization of the ferric hydroperoxo-active intermediate by preventing premature heterolytic O-O bond cleavage. This allows the enzyme to oxygenate selectively at the alpha-meso carbon in HmuO catalysis.

DOI： 10.1074/jbc.M311631200

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Language：Japanese   Publisher：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.43.S2_2

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE INC  

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Language：English   Publisher：AMER CHEMICAL SOC  

Alkyl-isocyanides are able to bind to both ferric and ferrous iran of the heme in cytochrome P450, and the resulting complexes exhibit characteristic optical absorption spectra. While the ferric complex gives a single Soret band at 430 nm, the ferrous complex shows double Soret bands at 430 and 450 nm. The ratio of intensities of the double Soret bands in the ferrous isocyanide complex of P450 varies, as a function of pH, ionic strength, and the origin of the enzyme. To understand the structural origin of these characteristic spectral features, we examined the crystallographic and spectrophotometric properties of the isocyanide complexes of Pseudomonas putida cytochrome P450cam and Fusarium oxysporum cytochorme P450nor, since ferrous isocyanide complex of P450cam gives a single Soret band at 453 nm, while that of P450nor gives one at 427 nm. Corresponding to the optical spectra, we observed C-N stretching of a ferrous iron-bound isocyanide at 2145 and 2116 cm(-1) for P450nor and P450cam, respectively. The crystal structures of the ferric and ferrous n-butyl isocyanide complexes of P450cam and P450nor were determined. The coordination structure of the fifth Cys thiolate was indistinguishable for the two P450s, but the coordination geometry of the isocyanide was different for the case of P450cam [d(Fe-C) = 1.86 Angstrom angle Fe-C-N = 159 degrees] versus P450nor [d(Fe-C)= 1.85 Angstrom, angle Fe-C-N = 175 degrees]. Another difference in the structures was the chemical environment of the heme pocket. In the case of P450cam, the iron-bound isocyanide is surrounded by some hydrophobic side chains, while, for P450nor, it is surrounded by polar groups including several water molecules. On the basis of these observations, we proposed that the steric factors and/or the polarity of the environment surrounding the iron-bound isocyanide significantly effect on the resonance structure of the heme(Fe)-isocyanide moiety and that differences in these two factors are responsible for the spectral characteristics for P450s.

DOI： 10.1021/bi002225s

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Language：English   Publisher：NATURE AMERICA INC  

Biliverdin reductase (BVR) is a soluble cytoplasmic enzyme that catalyzes the conversion of biliverdin to bilirubin using NADH or NADPH as electron donor. Bilirubin is a significant biological antioxidant, but it is also neurotoxic and the cause of kernicterus. In this study, we have determined the crystal structure of rat BVR at 1.4 Angstrom resolution. The structure contains two domains: an N-terminal domain characteristic of a dinucleotide binding fold (Rossmann fold) and a C-terminal domain that is predominantly an antiparallel six-stranded beta -sheet. Based on this structure, we propose modes of binding for NAD(P)H and biliverdin, and a possible mechanism for the enzyme.

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as a CO sensor. Cys(75) and His(77) are assigned to the fifth ligand of the ferric and ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis and EXAFS analyses to determine the coordination structure of the heme in CooA. Pro(2) is thought to be the sixth ligand of the ferric and ferrous hemes in CooA, which is consistent with the crystal structure of ferrous CooA (Lanzilotta, W, N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R, L,, Roberts, G. P., and Poulos, T. L. (2000) Not. Struct Biol. 7, 876-880). CooA exhibited anomalous redox chemistry, i.e. hysteresis was observed in electrochemical redox titrations in which the observed reduction and oxidation midpoint potentials were -320 mV and -260 mV, respectively. The redox-controlled ligand exchange of the heme between Cys(75) and His(77) is thought to cause the difference between the reduction and oxidation midpoint potentials.

DOI： 10.1074/jbc.M003972200

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Arg and Lys residues are concentrated on the distal side of cytochrome P450nor (P450nor) to form a positively charged cluster facing from the outside to the inside of the distal heme pocket. We constructed mutant proteins in which the Arg and Lys residues were replaced with Glu, Gin, or Ala. The results showed that this cluster plays crucial roles in NADH interaction. We also showed that some anions such as bromide (Br-) perturbed the heme environment along with the reduction step in P450nor-catalyzed reactions, which was similar to the effects caused by the mutations. We determined by x-ray crystallography that a Br-, termed an anion hole, occupies a key region neighboring heme, which is the terminus of the positively charged cluster and the terminus of the hydrogen bond network that acts as a proton delivery system, A comparison of the predicted mechanisms between the perturbations caused by Br- and the mutations suggested that Arg(174) and Arg(64) play a crucial role in binding NADH to the protein, These results indicated that the positively charged cluster is the unique structure of P450nor that responds to direct interaction with NADH.

DOI： 10.1074/jbc.M007244200

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DOI： 10.1006/jmbi.2000.4446

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Language：English   Publisher：MUNKSGAARD INT PUBL LTD  

The crystal structure of cytochrome c(6) from the red alga Porphyra yezoensis has been determined at 1.57 Angstrom resolution. The crystal is tetragonal and belongs to space group P4(3)2(1)2, with unit-cell parameters a = b = 49.26 (3), c = 83.45 (4) Angstrom and one molecule per asymmetric unit. The structure was solved by the molecular-replacement method and refined with X-PLOR to an R factor of 19.9% and a free R factor of 25.4%. The overall structure of cytochrome c6 follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys14 and Cys17, and the iron has an octahedral coordination with His18 and Met58 as the axial ligands. The sequence and the structure of the eukaryotic red algal cytochrome c6 are very similar to those of a prokaryotic cyanobacterial cytochrome c6 rather than those of eukaryotic green algal c6 cytochromes.

DOI： 10.1107/S090744490001461X

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Language：English   Publisher：ELSEVIER SCIENCE INC  

Threonine 243 of cytochrome P450nor (fungal nitric oxide reductase) corresponds to the 'conserved' Thr in the long I helix of monooxygenase cytochrome P450s. In P450nor, the replacement of Thr243 with Asn, Ala or Val makes the enzymatic activity dramatically reduce. In order to understand the roles of Thr243 in the reduction reaction of NO by P450nor, the crystal structures of three Thr243 mutants (Thr243--&gt;Asn, Thr243--&gt;Val, Thr243--&gt;Ala) of P450nor were determined at a 1.4-Angstrom resolution and at cryogenic temperature. However, the hydrogen-bonding pattern in the heme pocket of these mutants is essentially similar for that of the WT enzyme. This suggests that the determination of the structure of the NADH complex of P450nor is required, in order to evaluate the role of Thr243 in its enzymatic reaction. We attempted to crystallize the NADH complex under several conditions, but have not yet been successful. (C) 2000 Elsevier Science BN. All rights reserved.

DOI： 10.1016/S0162-0134(00)00161-6

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Language：Japanese  

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Language：English   Publisher：ACADEMIC PRESS LTD  

FixL of Rhizobium meliloti (RmFixL) is a sensor histidine kinase of the two-component system, which regulates the expression of the genes related to nitrogen fixation in the root nodule in response to the O-2 levels. The crystal structure of the sensor domain of FixL (RmFixLH), which contains a heme (Fe-porphyrin) as a sensing site, was determined at 1.4 Angstrom resolution. Based on the structural and spectroscopic analyses, we propose the O-2 sensing mechanism that differs from the case proposed in BjFixLH as follows; conformational changes in the F/G loop, which are induced by steric repulsion between the bent-bound O-2 and the Ile209 side-chain, would be transmitted to the histidine kinase domain. Interaction between the iron-bound O-2, and Ile209 was also observed in the resonance Raman spectra of RmFixLH as evidenced by the fact that the Fe-O-2 and Fe-CN stretching frequencies were shifted from 575 to 570 cm(-1) (Fe-O-2), and 504 to 499 cm(-1), respectively, as the result of the replacement of Ile209 with an Ala residue. In the I209A mutant of RmFixL, the O-2 sensing activity was destroyed, thus confirming our proposed mechanism. (C) 2000 Academic Press.

DOI： 10.1006/jmbi.2000.3954

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Language：English   Publisher：ELSEVIER SCIENCE INC  

Cytochrome P450nor (P450nor) is a heme enzyme isolated from the denitrifying fungus Fusarium oxysporum and catalyzes the NO reduction to N2O. Crystal structures of the wild type and two Ser286 mutants (Ser286--&gt;Val, Ser286--&gt;Thr) of P450nor have been determined for the ferric resting forms at a 1.7 Angstrom resolution at cryogenic temperature (100 K). We carried out three comparative analyses: (1) between the structures of P450nor at room temperature and cryogenic temperature, (2) between the structures of P450nor and four monooxygenase P450s, and (3) between the structures of the WT and the Ser286 mutant enzymes of P450nor. Comparison of the charge distribution on the protein surface suggests that proton and electron flow to the heme site is quite different in P450nor than in monooxygenase P450s. On the basis of the mutant structures, it was found that a special hydrogen-bonding network, Wat99-Ser286-Wat39-Asp393-solvent, acts as a proton delivery pathway in NO reduction by P450nor. In addition, the positively charged cluster located beneath the B'-helix is suggested as possible NADH binding site in P450nor, from which the direct two-electron transfer to the heme site allows to generate the characteristic intermediate in the NO reduction. These structural characteristics were not observed in structures of monooxygenase P450s, implying that these are factors determining the unique NO reduction activity of P450nor. (C) 2000 Elsevier Science S.A. All rights reserved.

DOI： 10.1016/S0162-0134(00)00103-3

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Language：Japanese   Publisher：日本結晶学会  

RIKEN beamline II (BL44B2) is dedicated to macromolecular crystallography in white and monochromatic X-ray mode, and XAFS of diluted biological systems. The user run was started since April 1998, and lots of scientific applications including time-resolved (intermediate state) protein crystallography, protein crystallography at atomic resolution, phasing with anomalous scattering, membrane protein crystallography, and fluorescence XAFS of diluted metalloproteins have been emerging.

DOI： 10.5940/jcrsj.42.106

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Fungal nitric-oxide reductase (NOR) is a heme enzyme that catalyzes the reduction of NO to N2O through its ferric-NO complex, the first intermediate of the catalysis. Crystal structures of the ferric-NO forms of wild type (WT) fungal NOR, and of the Ser(286) --&gt; Val and Ser(286) --&gt; Thr mutant enzymes were determined to 1.7-Angstrom resolution at cryogenic temperature (100 K). This shows a slightly tilted and bent NO binding to the heme iron, in sharp contrast to the highly bent NO coordination found in ferrous hemoproteins, In the WT structure, a specific hydrogen-bonding network that connects the active site to the solvent was identified, H2O(Wat(74))-Ser(286)-H2O- (Wat(33))-Asp(393)-solvent. Wat(74) is located 3.10 Angstrom from the iron-bound NO. Replacement of Ser(286) With Val or Thr scarcely alters the NO coordination structure but expels the water molecules, Wat(74) from the active site. The Asp(393) mutation does not influence the position of Wat74 but disrupts the hydrogen-bonding network at Wat(33), as evidenced by enzymatic, kinetic, and spectroscopic (resonance Raman and IR) results. The structural changes observed upon the Ser(286) or the Asp(393) mutation are consistent with the dramatic loss of the enzymatic activity for the NO reduction of fungal NOR. We have conclusively identified the water molecule, Wat(74), adjacent to the iron-bound NO as a proton donor to the Fe-NO moiety. In addition, we find the hydrogen-bonding network, H2O(Wat(74))-Ser-(286)-H2O(Wat(33))-Asp(393), as a proton delivery pathway in the NO reduction reaction by fungal NOR.

DOI： 10.1074/jbc.275.7.4816

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Language：Japanese   Publisher：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.40.S122_3

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Language：English   Publisher：ACADEMIC PRESS LTD  

Studies of oxygen equilibrium properties of Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrid hemoglobins (i.e. alpha(2)(Fe)beta(2)(M) and alpha(2)(M)beta(2)(Fe); M = Mg(II),Zn(II) (neither of these closed-shell metal ions binds oxygen or carbon monoxide)) are reported along with the X-ray crystal structures of alpha(2)(Fe)beta(2)(Mg) with and without CO bound. We found that Mg(II)-Fe(II) hybrids resemble Zn(II)-Fe(II) hybrids very closely in oxygen equilibrium properties. The Fe(II)-subunits in these hybrids bind oxygen with very low affinities, and the effect of allosteric effecters, such as proton and/or inositol hexaphosphate, is relatively small. We also found a striking similarity in spectrophotometric properties between Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrids, particularly, the large spectral changes that occur specifically in the metal-containing beta subunits upon the R-T transition of the hybrids. In crystals, both alpha(2)(Fe)beta(2)(Mg) and alpha(2)(Fe-CO)beta(2)(Mg) adopt the quaternary structure of deoxyhemoglobin. These results, combined with the re-evaluation of the oxygen equilibrium properties of normal hemoglobin, low-affinity mutants, and metal substituted hybrids, point to a general tendency of human hemoglobin that when the association equilibrium constant of hemoglobin for the first binding oxygen molecule (K-1) approaches 0.004 mmHg(-1), the cooperativity as well as the effect of allosteric effecters is virtually abolished. This is indicative of the existence of a distinct thermodynamic state which determines the lowest oxygen affinity of human hemoglobin. Moreover, excellent agreement between the reported oxygen affinity of deoxyhemoglobin in crystals and the lowest affinity in solution leads us to propose that the classical T structure of deoxyhemoglobin in the crystals represents the lowest affinity state in solution.
We also survey the oxygen equilibrium properties of various metal-substituted hybrid hemoglobins studied over the past 20 years in our laboratory. The bulk of these data are consistent with the Perutz's trigger mechanism, in that the affinity of a metal hybrid is determined by the ionic radius of the metal, and also by the steric effect of the distal ligand, if present. However, there remains a fundamental contradiction among the oxygen equilibrium properties of the beta substituted hybrid hemoglobins. (C) 1999 Academic Press.

DOI： 10.1006/jmbi.1999.3124

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Language：English   Publisher：ACADEMIC PRESS INC  

Hmu O is a 24-kDa soluble bacterial heme degradation enzyme found in the pathogen Corynebacterium diphtheriae, the causative agent of diphtheria. Similar to the mammalian heme oxygenase, it binds hemin stoichiometrically and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Iron is an essential nutrient for bacteria and especially important for pathogenesis. Here we report the first crystallization and preliminary crystallographic study of the heme-Hmu O complex formed from hemin and a recombinant Hmu O, which was expressed in Escherichia coli from a synthetic gene based on the putative hmu O gene sequence. Crystals of the heme Hmu O complex were obtained by the sitting drop vapor diffusion method using a precipitant solution containing 18% (w/v) PEG 8000 and 0.2 M calcium acetate in 0.1 M sodium cacodylate (pH 6.5). Using synchrotron radiation, the heme-Hmu O crystal diffracted to 2.8 Angstrom resolution. It belongs to the monoclinic space group C2, with unit cell parameters a = 123.18 Angstrom, b = 44.51 Angstrom, c = 92.10 Angstrom, and beta = 123.3 degrees. Assuming one molecule of the heme-Hmu O complex per asymmetric unit, the calculated value of V-m is 2.89 Angstrom(3)/Da. (C) 1999 Academic Press.

DOI： 10.1006/jsbi.1999.4122

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PubMed

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Language：Japanese   Publisher：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.39.S105_4

CiNii Books

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Language：Japanese   Publisher：一般社団法人日本生物物理学会  

CiNii Books

J-GLOBAL

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Language：English   Publisher：NATURE PUBLISHING CO  

Structures of nitric oxide reductase (NOR) in the ferric resting and the ferrous CO states have been solved at 2.0 Angstrom resolution. These structures provide significant new insights into how NO is reduced in biological systems, The haem distal pocket is open to solvent, implicating this region as a possible NADH binding site, In combination with mutagenesis results, a hydrogen-bonding network from the water molecule adjacent to the iron ligand to the protein surface of the distal pocket through the hydroxyl group of Ser 286 and the carboxyl group of Asp 393 can be assigned to a pathway for proton delivery during the NO reduction reaction.

DOI： 10.1038/nsb1097-827

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：FEDERATION AMER SOC EXP BIOL  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：FEDERATION AMER SOC EXP BIOL  

Web of Science

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Language：English  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (international conference proceedings)   Publisher：INT UNION CRYSTALLOGRAPHY  

DOI： 10.1107/S0108767396093762

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