記述言語：英語   掲載種別：研究論文（学術雑誌）  

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ into the endoplasmic reticulum (ER). Herein, we present cryo-electron microscopy (EM) structures of three intermediates of SERCA2b: Ca2+-bound phosphorylated (E1P·2Ca2+) and Ca2+-unbound dephosphorylated (E2·Pi) intermediates and another between the E2P and E2·Pi states. Our cryo-EM analysis demonstrates that the E1P·2Ca2+ state exists in low abundance and preferentially transitions to an E2P-like structure by releasing Ca2+ and that the Ca2+ release gate subsequently undergoes stepwise closure during the dephosphorylation processes. Importantly, each intermediate adopts multiple sub-state structures including those like the next one in the catalytic series, indicating conformational overlap at transition steps, as further substantiated by atomistic molecular dynamic simulations of SERCA2b in a lipid bilayer. The present findings provide insight into how enzymes accelerate catalytic cycles.

DOI： 10.1016/j.celrep.2022.111760

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) 2b is a ubiquitous SERCA family member that conducts Ca2+ uptake from the cytosol to the ER. Herein, we present a 3.3 Å resolution cryo-electron microscopy (cryo-EM) structure of human SERCA2b in the E1·2Ca2+ state, revealing a new conformation for Ca2+ -bound SERCA2b with a much closer arrangement of cytosolic domains than in the previously reported crystal structure of Ca2+ -bound SERCA1a. Multiple conformations generated by 3D classification of cryo-EM maps reflect the intrinsically dynamic nature of the cytosolic domains in this state. Notably, ATP binding residues of SERCA2b in the E1·2Ca2+ state are located at similar positions to those in the E1·2Ca2+ -ATP state; hence, the cryo-EM structure likely represents a preformed state immediately prior to ATP binding. Consistently, a SERCA2b mutant with an interdomain disulfide bridge that locks the closed cytosolic domain arrangement displayed significant autophosphorylation activity in the presence of Ca2+ . We propose a novel mechanism of ATP binding to SERCA2b.

DOI： 10.15252/embj.2021108482

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo-electron microscopy (cryo-EM) structures of human SERCA2b in E1∙2Ca2+-adenylyl methylenediphosphonate (AMPPCP) and E2-BeF3- states at 2.9- and 2.8-Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7 and approaches the luminal loop flanked by TM7 and TM8. While the LE served to stabilize the cytosolic and TM domain arrangement of SERCA2b, deletion of the LE rendered the overall conformation resemble that of SERCA1a and SERCA2a and allowed multiple conformations. Thus, the LE appears to play a critical role in conformational regulation in SERCA2b, which likely explains the different kinetic properties of SERCA2b from those of other isoforms lacking the LE.

DOI： 10.1126/sciadv.abb0147

記述言語：英語  

Conspectus[NiFe] hydrogenases catalyze reversible hydrogen production/consumption. The core unit of [NiFe] hydrogenase consists of a large and a small subunit. The active site of the large subunit of [NiFe] hydrogenases contains a NiFe(CN)2CO cluster. The biosynthesis/maturation of these hydrogenases is a complex and dynamic process catalyzed primarily by six Hyp proteins (HypABCDEF), which play central roles in the maturation process. HypA and HypB are involved in the Ni insertion, whereas HypC, D, E, and F are required for the biosynthesis, assembly, and insertion of the Fe(CN)2CO group. HypE and HypF catalyze the synthesis of the CN group through the carbamoylation and cyanation of the C-terminus cysteine of HypE. HypC and HypD form a scaffold for the assembly of the Fe(CN)2CO moiety.Over the last decades, a large number of biochemical studies on maturation proteins have been performed, revealing basic functions of each Hyp protein and the overall framework of the maturation pathway. However, it is only in the last 10 years that structural insight has been gained, and our group has made significant contributions to the structural biology of hydrogenase maturation proteins.Since our first publication, where crystal structures of three Hyp proteins have been determined, we have performed a series of structural studies of all six Hyp proteins from a hyperthermophilic archaeon Thermococcus kodakarensis, providing molecular details of each Hyp protein. We have also determined the crystal structures of transient complexes between Hyp proteins that are formed during the maturation process to sequentially incorporate the components of the NiFe(CN)2CO cluster to immature large subunits of [NiFe] hydrogenases. Such complexes, whose crystal structures are determined, include HypA-HypB, HypA-HyhL (hydrogenase large subunit), HypC-HypD, and HypC-HypD-HypE. The structures of the HypC-HypD, and HypCDE complexes reveal a sophisticated process of transient formation of the HypCDE complex, providing insight into the molecular basis of Fe atom cyanation. The high-resolution structures of the carbamoylated and cyanated forms of HypE reveal a structural basis for the biological conversion of primary amide to nitrile. The structure of the HypA-HypB complex elucidates nucleotide-dependent transient complex formation between these two proteins and the molecular basis of acquisition and release of labile Ni. Furthermore, our recent structure analysis of a complex between HypA and immature HyhL reveals that spatial rearrangement of both the N- and C-terminal tails of HyhL will occur upon the [NiFe] cluster insertion, which function as a key checkpoint for the maturation completion. This Account will focus on recent advances in structural studies of the Hyp proteins and on mechanistic insights into the [NiFe] hydrogenase maturation.

DOI： 10.1021/acs.accounts.0c00022

記述言語：英語   掲載種別：研究論文（学術雑誌）  

High-Resolution Crystal Structure of Arabidopsis FLOWERING LOCUS T Illuminates Its Phospholipid-Binding Site in Flowering.
Arabidopsis FLOWERING LOCUS T (FT) is a pivotal component of florigen, a long-range mobile flowering signal. Here, we determined the 1.0 Å-resolution crystal structure of FT, a significantly higher-resolution crystal structure of FT than previously reported one (2.6 Å). The present crystallographic studies revealed 4 alternative configurations with the precise location of the surrounding water molecules. Using this structural data, computational docking simulation predicted the putative binding sites for phosphatidylcholine (PC), an endogenous ligand that interacts with FT to modulate flowering time. In vitro reconstitution of the lipid-protein interaction showed that mutations at two of the predicted sites significantly compromised the lipid binding ability of FT. In planta, one of the mutant FT proteins significantly affected FT function in flowering, emphasizing the involvement of PC binding in modulating FT function. Our structural, biochemical, and transgenic analyses reveal the molecular mechanism of PC binding in FT-mediated flowering time control.

DOI： 10.1016/j.isci.2019.10.045

記述言語：その他   掲載種別：研究論文（学術雑誌）  

Structural Basis of Sarco/Endoplasmic Reticulum Ca²⁺-ATPase 2b Regulation via Transmembrane Helix Interplay.

DOI： 10.1016/j.celrep.2019.03.106

記述言語：その他   掲載種別：研究論文（学術雑誌）  

Zinc regulates ERp44-dependent protein quality control in the early secretory pathway

DOI： 10.1038/s41467-019-08429-1

記述言語：その他   掲載種別：研究論文（学術雑誌）  

Crystal structures of a [NiFe] hydrogenase large subunit HyhL in an immature state in complex with a Ni chaperone HypA.
Ni-Fe clusters are inserted into the large subunit of [NiFe] hydrogenases by maturation proteins such as the Ni chaperone HypA via an unknown mechanism. We determined crystal structures of an immature large subunit HyhL complexed with HypA from <italic>Thermococcus kodakarensis</italic>. Structure analysis revealed that the N-terminal region of HyhL extends outwards and interacts with the Ni-binding domain of HypA. Intriguingly, the C-terminal extension of immature HyhL, which is cleaved in the mature form, adopts a β-strand adjacent to its N-terminal β-strands. The position of the C-terminal extension corresponds to that of the N-terminal extension of a mature large subunit, preventing the access of endopeptidases to the cleavage site of HyhL. These findings suggest that Ni insertion into the active site induces spatial rearrangement of both the N- and C-terminal tails of HyhL, which function as a key checkpoint for the completion of the Ni-Fe cluster assembly.

DOI： 10.1073/pnas.1801955115

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate L-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

DOI： 10.1038/s41467-017-01311-y

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ERdj5, composed of an N-terminal J domain followed by six thioredoxin-like domains, is the largest protein disulfide isomerase family member and functions as an ER-localized disulfide reductase that enhances ER-associated degradation (ERAD). Our previous studies indicated that ERdj5 comprises two regions, the N- and C-terminal clusters, separated by a linker loop and with distinct functional roles in ERAD. We here present a new crystal structure of ERdj5 with a largely different cluster arrangement relative to that in the original crystal structure. Single-molecule observation by high-speed atomic force microscopy visualized rapid cluster movement around the flexible linker loop, indicating the highly dynamic nature of ERdj5 in solution. ERdj5 mutants with a fixed-cluster orientation compromised the ERAD enhancement activity, likely because of less efficient reduction of aberrantly formed disulfide bonds and prevented substrate transfer in the ERdj5-mediated ERAD pathway. We propose a significant role of ERdj5 conformational dynamics in ERAD of disulfide-linked oligomers.

DOI： 10.1016/j.str.2017.04.001

記述言語：その他   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/ange.201701654

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7U(A), C7U(B)] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A-and B-chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se-Ins with IDE was significantly decelerated (t(1/2) approximate to 8 h vs. approximate to 1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.

DOI： 10.1002/anie.201701654

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ERp44 retrieves some endoplasmic reticulum (ER)-resident enzymes and immature oligomers of secretory proteins from the Golgi. Association of ERp44 with its clients is regulated by pH-dependent mechanisms, but the molecular details are not fully understood. Here we report high-resolution crystal structures of human ERp44 at neutral and weakly acidic pH. These structures reveal key regions in the C-terminal tail (C tail) missing in the original crystal structure, including a regulatory histidine-rich region and a subsequent extended loop. The former region forms a short alpha-helix (alpha 16), generating a histidine-clustered site (His cluster). At low pH, the three Trx-like domains of ERp44 ("a," "b," and "b'") undergo significant rearrangements, likely induced by protonation of His157 located at the interface between the a and b domains. The alpha 16-helix is partially unwound and the extended loop is disordered in weakly acidic conditions, probably due to electrostatic repulsion between the protonated histidines in the His cluster. Molecular dynamics simulations indicated that helix unwinding enhances the flexibility of the C tail, disrupting its normal hydrogen-bonding pattern. The observed pH-dependent conformational changes significantly enlarge the positively charged regions around the client-binding site of ERp44 at low pH. Mutational analyses showed that ERp44 forms mixed disulfides with specific cysteines residing on negatively charged loop regions of Ero1 alpha. We propose that the protonation states of the essential histidines regulate the ERp44-client interaction by altering the C-tail dynamics and surface electrostatic potential of ERp44.

DOI： 10.1073/pnas.1621426114

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Calcium ion (Ca2+) is an important second messenger that regulates numerous cellular functions. Intracellular Ca2+ concentration ([Ca2+](i)) is strictly controlled by Ca2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca2+ from the cytosol into the ER in an ATPase activity-dependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond. Notably, ERdj5 activated SERCA2b at a lower ER luminal [Ca2+] ([Ca2+](ER)), whereas a higher [Ca2+](ER) induced ERdj5 to form oligomers that were no longer able to interact with the pump, suggesting [Ca2+](ER)-dependent regulation. Binding Ig protein, an ER-resident molecular chaperone, exerted a regulatory role in the oligomerization by binding to the J domain of ERdj5. These results identify ERdj5 as one of the master regulators of ER calcium homeostasis and thus shed light on the importance of cross talk among redox, Ca2+, and protein homeostasis in the ER.

DOI： 10.1073/pnas.1605818113

記述言語：英語   掲載種別：研究論文（学術雑誌）  

A [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1 (TkHybD) is involved in the cleavage of the C-terminal residues of [NiFe] hydrogenase large subunits by Ni recognition. Here, we report the crystal structure of TkHybD at 1.82 angstrom resolution to better understand this process. TkHybD exhibits an // sandwich fold with conserved residues responsible for the Ni recognition. Comparisons of TkHybD with homologous proteins also reveal that they share a common overall architecture, suggesting that they have similar catalytic functions. Our results including metal binding site prediction provide insight into the substrate recognition and catalysis mechanism of TkHybD. Proteins 2016; 84:1321-1327. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/prot.25070

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypB(AT)) with Ni ions. Transient association between HypA and HypB(AT) is controlled by the ATP hydrolysis cycle of HypB(AT), which is accelerated by HypA. Only the ATP-bound form of HypB(AT) can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypAB(AT) complex has a nanomolar affinity (K-d = 7 nM), which is in contrast to the micromolar affinity (Kd = 4 mu M) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypB(AT), affecting its association with HypA. These findings indicate that HypA and HypB(AT) constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypB(AT) functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.

DOI： 10.1073/pnas.1503102112

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hydrogenase pleiotropically acting protein (Hyp)E plays a role in biosynthesis of the cyano groups for the NiFe(CN)2CO center of [NiFe] hydrogenases by catalyzing the ATP-dependent dehydration of the carbamoylated C-terminal cysteine of HypE to thiocyanate. Although structures of HypE proteins have been determined, until now there has been no structural evidence to explain how HypE dehydrates thiocarboxamide into thiocyanate. Here, we report the crystal structures of the carbamoylated and cyanated forms of HypE from Thermococcus kodakarensis in complex with nucleotides at 1.53- and 1.64-Å resolution, respectively. Carbamoylation of the C-terminal cysteine (Cys338) of HypE by chemical modification is clearly observed in the present structures. In the presence of ATP, the thiocarboxamide of Cys338 is successfully dehydrated into the thiocyanate. In the carbamoylated state, the thiocarboxamide nitrogen atom of Cys338 is close to a conserved glutamate residue (Glu272), but the spatial position of Glu272 is less favorable for proton abstraction. On the other hand, the thiocarboxamide oxygen atom of Cys338 interacts with a conserved lysine residue (Lys134) through a water molecule. The close contact of Lys134 with an arginine residue lowers the pKa of Lys134, suggesting that Lys134 functions as a proton acceptor. These observations suggest that the dehydration of thiocarboxamide into thiocyanate is catalyzed by a two-step deprotonation process, in which Lys134 and Glu272 function as the first and second bases, respectively.

DOI： 10.1073/pnas.1313620110

記述言語：英語   掲載種別：研究論文（学術雑誌）  

HypB (metal-binding GTPase) and HypA (nickel metallochaperone) are required for nickel insertion into [NiFe] hydrogenase. However, the HypB homolog proteins are not found in some archaeal species including Thermococcales. In this article, we Identify a novel archaeal Mrp/MinD family ATPase-type HypB from Thermococcus kodakarensis (Tk-mmHypB) and determine its crystal structure. The mmhypB gene is conserved among species lacking the hypB gene and is located adjacent to the hypA gene on their genome. Deletion of the mmhypB gene leads to a significant reduction in hydrogen-dependent growth of T. kodakarensis, which is restored by nickel supplementation. The monomer structure of Tk-mmHypB is similar to those of the Mrp/MinD family ATPases. The ADP molecules are tightly bound to the protein. Isothermal titration calorimetry shows that Tk-mmHypB binds ATP with a K-d value of 84 nM. ADP binds more tightly than does ATP, with a K-d value of 15 nM. The closed Tk-mmHypB dimer in the crystallographic asymmetric unit is consistent with the ATP-hydrolysis-deficient dimer of the Mrp/MinD family Soj/MinD proteins. Structural comparisons with these proteins suggest the ATP-binding dependent conformational change and rearrangement of the Tk-mmHypB dimer. These observations imply that the nickel insertion process during the [NiFe] hydrogenase maturation is performed by HypA, mmHypB, and a nucleotide exchange factor in these archaea. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2013.02.004

記述言語：英語   掲載種別：研究論文（学術雑誌）  

[NiFe] hydrogenase maturation represents one of the most dynamic and sophisticated processes in metallocenter assembly. The Fe(CN)(2)CO moiety of [NiFe] hydrogenases is assembled via unknown transient interactions among specific maturation proteins HypC (metallochaperone), HypD (redox protein), and HypE (cyanide synthesis/donor). Here, we report the structures of the HypC-HypD and HypC-HypD-HypE complexes, providing a view of the transient interactions that take place during the maturation process. HypC binds to the conserved region of HypD through extensive hydrophobic interactions. The ternary complex formation between HypE and the HypCD complex involves both HypC and HypD, rendering the HypE conformation favorable for cyanide transfer. In the complex, the conserved cysteines of HypC and HypD form an Fe binding site. The conserved C-terminal cysteine of HypE can access the thiol redox cascade of HypD. These results provide structural insights into the Fe atom cyanation in the HypCDE complex.

DOI： 10.1016/j.str.2012.09.018

記述言語：英語   掲載種別：研究論文（学術雑誌）  

HypF is involved in the biosynthesis of the CN ligand of the NiFe(CN)(2)CO centre of [NiFe]-hydrogenases. Here, the full-length structure of HypF from Thermococcus kodakarenesis is reported at 4.5 angstrom resolution. The N-terminal acylphosphatase-like (ACP) domain interacts with the zinc-finger domain with some flexibility in its relative position. Molecular-surface analysis shows that a deep pocket formed between the ACP and zinc-finger domains is highly conserved and has positive potential. These results suggest that the positively charged pocket identified is involved in the hydrolysis of carbamoyl phosphate and the formation of a carbamoyl intermediate.

DOI： 10.1107/S1744309112036421

記述言語：英語  

[NiFe] hydrogenases catalyze reversible hydrogen production/consumption. The active site of [NiFe] hydrogenases contains a complex NiFe(CN)(2)CO center, and the biosynthesis/maturation of these enzymes is a complex and dynamic process, primarily involving six Hyp proteins (HypABCDEF). HypA and HypB are involved in the Ni insertion, whereas the other four Hyp proteins (HypCDEF) are required for the biosynthesis, assembly and insertion of the Fe(CN)(2)CO group. Over the last decades, a large number of functional and structural studies on maturation proteins have been performed, revealing detailed functions of each Hyp protein and the framework of the maturation pathway. This article will focus on recent advances in structural studies of the Hyp proteins and on mechanistic insights into the [NiFe] hydrogenase maturation.

DOI： 10.1515/hsz-2012-0197

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The large subunit of the [NiFe] hydrogenases harbors a NiFe(CN)(2)(CO) cluster. Maturation proteins HypA, B, C, D, E, and F are required for the NiFe cluster biosynthesis. While the maturation machinery has been hitherto studied intensively, little is known about interactions between the Hyp proteins and the large subunit of the [NiFe] hydrogenase. In this study, we have purified and characterized the cytosolic [NiFe] hydrogenase large subunit HyhL from Thermococcus kodakarensis (Tk-HyhL). Tk-HyhL exists in equilibrium between monomeric and dimeric forms. In vitro interaction analyses showed that Tk-HyhL monomer forms a tight complex with Tk-HypA and weakly interacts with Tk-HypC. The expected ternary complex formation was not detected. These observations reflect a diversity in the mechanism of Ni insertion in [NiFe] hydrogenase maturation depending on the organism. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.11.083

記述言語：英語   掲載種別：研究論文（学術雑誌）  

HypA is one of the auxiliary proteins involved in the maturation of [NiFe] hydrogenases. By an unknown mechanism, HypA functions as a metallochaperone in the insertion of the Ni atom into hydrogenases. We have determined the crystal structures of HypA from Thermococcus kodakaraensis KOD1 in both monomeric and dimeric states. The structure of the HypA monomer consists of Ni- and Zn-binding domains. The relative arrangement of the two metal-binding domains has been shown to be associated with local conformations of the conserved Ni-binding motif, suggesting a communication between the Ni- and Zn-binding sites. The HypA dimer has been shown to be stabilized by unexpected domain swapping through archaea-specific linker helices. In addition, the hexameric structure of HypA is formed in the crystal packing. Several hydrogen bonds and hydrophobic interactions stabilize the hexamer interface. These findings suggest the functional diversity of HypA proteins. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2009.09.030

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The [2Fe-2S] transcription factor SoxR, a member of the MerR family, functions as a bacterial sensor of oxidative stress such as superoxide and nitric oxide. SoxR is activated by reversible one-electron oxidation of the [2Fe-2S] cluster and then enhances the production of various antioxiclant. proteins through the soxRS regulon. In the active state, SoxR and other MerR family proteins activate transcription from unique promoters, which have a long 19- or 20-bp spacer between the -35 and -10 operator elements, by untwisting the promoter DNA. Here, we show the crystal structures of SoxR and its complex with the target promoter in the oxidized (active) state. The structures reveal that the [2Fe-2S] cluster of SoxR is completely solvent-exposed and surrounded by an asymmetric environment stabilized by interaction with the other subunit. The asymmetrically charged environment of the [2Fe-2S] cluster probably causes redox-dependent conformational changes of SoxR and the target promoter. Compared with the promoter structures with the 19-bp spacer previously studied, the DNA structure is more sharply bent, by approximate to 1 bp, with the two central base pairs holding Watson-Crick base pairs. Comparison of the target promoter sequences of the MerR family indicates that the present DNA structure represents the activated conformation of the target promoter with a 20-bp spacer in the MerR family.

DOI： 10.1073/pnas.0709188105

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The hydrogenase maturation protein HypE is involved in the biosynthesis of the CN ligands of the active-site iron of [ NiFe] hydrogenases using carbamoylphosphate as a substrate. Here, the crystallization and preliminary crystallographic analysis of HypE from Thermococcus kodakaraensis KOD1 are reported. Crystals of HypE ( 338 amino acids, 35.9 kDa) have been obtained by the sitting-drop vapour-diffusion method using 2-methyl-2,4-pentanediol ( MPD) as a precipitant. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 88.3, b = 45.8, c = 75.1 angstrom. There is one HypE molecule in the asymmetric unit. A complete native X-ray diffraction data set was collected to a maximum resolution of 1.55 angstrom at 100 K.

DOI： 10.1107/S1744309107038833

記述言語：英語   掲載種別：研究論文（学術雑誌）  

[NiFe] hydrogenase maturation proteins HypC, HypD, and HypE catalyze the insertion and cyanation of the iron center of [NiFe] hydrogenases by an unknown mechanism. We have determined the crystal structures of HypC, HypD, and HypE from Thermococcus kodakaraensis KOD1 at 1.8 angstrom, 2.07 angstrom, and 1.55 angstrom resolution, respectively. The structure of HypD reveals its probable iron binding and active sites for cyanation. An extended conformation of each conserved motif of HypC and HypE allows the essential cysteine residues of both proteins to interact with the active site of HypD. Furthermore, the C-terminal tail of HypE is shown to exist in an ATP-dependent dynamic equilibrium between outward and inward conformations. Unexpectedly, the [4Fe-4S] cluster environment of HypD is quite similar to that of ferredoxin: thioredoxin reductase (FTR), indicating the existence of a redox cascade similar to the FTR system. These results suggest a cyanation reaction mechanism via unique thiol redox signaling in the HypCDE complex.

DOI： 10.1016/j.molcel.2007.05.039

記述言語：英語   掲載種別：研究論文（学術雑誌）  

HypC and HypD proteins are required for the insertion of the Fe atom with diatomic ligands into the large subunit of [ NiFe] hydrogenases, an important step in the maturation process of this type of hydrogenase. The crystallization and preliminary crystallographic analysis of HypC and HypD from Thermococcus kodakaraensis KOD1 are reported. Crystals of HypC grew in two different forms. Monoclinic crystals of HypC in space group C2 with unit-cell parameters a = 78.2, b = 59.1, c = 54.0 angstrom, beta = 109.0 degrees were obtained using PEG 4000 and ammonium sulfate or sodium bromide as precipitants. They diffracted X-rays to 1.8 angstrom resolution and were suitable for structure determination. Crystals of HypD were also obtained in two different forms. The monoclinic crystals obtained using PEG 4000 and magnesium chloride diffracted X-rays to beyond 2.1 angstrom resolution, despite growing as clusters. They belong to space group P2(1), with unit-cell parameters a = 42.3, b = 118.4, c = 81.2 angstrom, beta = 100.9 degrees, and are suitable for data collection.

DOI： 10.1107/S1744309107023391

記述言語：英語   掲載種別：研究論文（学術雑誌）  

SoxR, a member of the MerR family of transcriptional activators, functions as a sensor of oxidative stress. The redox states of the 2Fe-2S cluster of SoxR regulate the activity of SoxR. Here, the crystallization and preliminary crystallographic analysis of SoxR and its complex with DNA are reported. Crystals of SoxR were obtained using PEG 10 000 and glycerol as precipitants. The crystals of SoxR belong to space group P6(2) or P6(4), with unit-cell parameters a = b = 80.0, c = 88.1 angstrom. Crystals of the SoxR-DNA complex were obtained using a 20 bp DNA fragment from a condition containing PEG 10 000 and sodium/potassium tartrate. The crystals of the SoxR-DNA complex belong to space group P6122 or P6522, with unit-cell parameters a = b = 53.5, c = 355.6 angstrom. Diffraction data were collected to a maximum resolution of 3.2 and 2.7 angstrom for SoxR and the SoxR-DNA complex, respectively.

DOI： 10.1107/S1744309106048482

記述言語：英語   掲載種別：研究論文（学術雑誌）  

SufC, a cytoplasmic ABC-ATPase, is one of the most conserved Suf proteins. SufC forms a stable complex with SufB and SufD, and the SufBCD complex interacts with other Suf proteins in the Fe-S cluster assembly. We have determined the crystal structure of SufC from Thermus.thermophilus HB8 in nucleotide-free and ADP-Mg-bound states at 1.7 angstrom and 1.9 angstrom resolution, respectively. The overall architecture of the SufC structure is similar to other ABC ATPases structures, but there are several specific motifs in SufC. Three residues following the end of the Walker B motif form a novel 3(10) helix which is not observed in other ABC ATPases. Due to the novel 3(10) helix, a conserved glutamate residue involved in ATP hydrolysis is flipped out. Although this unusual conformation is unfavorable for ATP hydrolysis, salt-bridges formed by conserved residues and a strong hydrogen-bonding network around the novel 3(10) helix suggest that the novel 3(10) helix of SufC is a rigid conserved motif. Compared to other ABC-ATPase structures, a significant displacement occurs at a linker region between the ABC alpha/beta domain and the alpha-helical domain. The linker conformation is stabilized by a hydrophobic interaction between conserved residues around the Q loop. The molecular surfaces of SufC and the C-terminal helices of SufD (PDB code: 1VH4) suggest that the unusual linker conformation conserved among SufC proteins is probably suitable for interacting with Suf beta and SufD. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jmb.2005.09.017

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Structural basis of zinc-dependent protein quality control in the early secretory pathway

記述言語：日本語  

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Structural study of Hyp protein complexes for the maturation of [NiFe] hydrogenase from Thermococcus kodakarensis

記述言語：日本語  

[NiFe]ヒドロゲナーゼへNiを組み込むHypAB複合体の結晶構造解析

記述言語：日本語  

[NiFe]ヒドロゲナーゼ成熟化おけるシアノ基生合成中間体の構造解析

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Crystal Structure of the Oxidative-Stress Sensor SoxR Bound to DNA

記述言語：日本語  

[NiFe]ヒドロゲナーゼは，水素分子の可逆的な酸化還元反応を触媒する酵素で，活性部位にはシアノ基と一酸化炭素基を配位子にもつNiとFe原子の金属クラスターがある．この複雑な金属クラスターは，生体内で自発的につくられるのではなく，成熟化因子と呼ばれる特異的なタンパク質，Hypタンパク質群の助けによって，段階的に組み込まれて生合成される．最近，Hypタンパク質の立体構造が相次いで決定されて，[NiFe]ヒドロゲナーゼ成熟化機構についての構造生物学的研究は，新しい進展を見せている．

DOI： 10.1271/kagakutoseibutsu.46.608

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