Kunio Miki

Find an error

Name:

Organization: Kyoto University , Japan

Department: Department of Chemistry

Title: Professor(PhD)

TOPICS

Inorganic Chemistry

hydrogenase models

Chemicals
References

Crystal structure of ketopantoate reductase from Thermococcus kodakarensis complexed with NADP+

Co-reporter:Yoshiki Aikawa;Yuichi Nishitani;Hiroya Tomita;Haruyuki Atomi

Acta Crystallographica Section F 2016 Volume 72( Issue 5) pp:369-375

Publication Date(Web):

DOI:10.1107/S2053230X16005033

Coenzyme A (CoA) plays pivotal roles in a variety of metabolic pathways in all organisms. The biosynthetic pathway of CoA is strictly regulated by feedback inhibition. In the hyperthermophilic archaeon Thermococcus kodakarensis, ketopantoate reductase (KPR), which catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate, is a target of feedback inhibition by CoA. The crystal structure of KPR from T. kodakarensis (Tk-KPR) complexed with CoA and 2-oxopantoate has previously been reported. The structure provided an explanation for the competitive inhibition mechanism. Here, further biochemical analyses of Tk-KPR and the crystal structure of Tk-KPR in complex with NADP⁺ are reported. A mutational analysis implies that the residues in the binding pocket cooperatively contribute to the recognition of CoA. The structure reveals the same dimer architecture as the Tk-KPR–CoA–2-oxopantoate complex. Moreover, the positions of the residues involved in the dimer interaction are not changed by the binding of CoA and 2-oxopantoate, suggesting individual conformational changes of Tk-KPR monomers.

Crystal structure of the TK2203 protein from Thermococcus kodakarensis, a putative extradiol dioxygenase

Co-reporter:Yuichi Nishitani;Jan-Robert Simons;Tamotsu Kanai;Haruyuki Atomi

Acta Crystallographica Section F 2016 Volume 72( Issue 6) pp:427-433

Publication Date(Web):

DOI:10.1107/S2053230X16006920

The TK2203 protein from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (262 residues, 29 kDa) is a putative extradiol dioxygenase catalyzing the cleavage of C–C bonds in catechol derivatives. It contains three metal-binding residues, but has no significant sequence similarity to proteins for which structures have been determined. Here, the first crystal structure of the TK2203 protein was determined at 1.41 Å resolution to investigate its functional role. Structure analysis reveals that this protein shares the same fold and catalytic residues as other extradiol dioxygenases, strongly suggesting the same enzymatic activity. Furthermore, the important region contributing to substrate selectivity is discussed.

Crystal Structure and Product Analysis of an Archaeal myo-Inositol Kinase Reveal Substrate Recognition Mode and 3-OH Phosphorylation

Co-reporter:Ryuhei Nagata, Masahiro Fujihashi, Takaaki Sato, Haruyuki Atomi, and Kunio Miki

Biochemistry 2015 Volume 54(Issue 22) pp:3494-3503

Publication Date(Web):May 14, 2015

DOI:10.1021/acs.biochem.5b00296

The TK2285 protein from Thermococcus kodakarensis was recently characterized as an enzyme catalyzing the phosphorylation of myo-inositol. Only two myo-inositol kinases have been identified so far, the TK2285 protein and Lpa3 from Zea mays, both of which belong to the ribokinase family. In either case, which of the six hydroxyl groups of myo-inositol is phosphorylated is still unknown. In addition, little is known about the myo-inositol binding mechanism of these enzymes. In this work, we determined two crystal structures: those of the TK2285 protein complexed with the substrates (ATP analogue and myo-inositol) or the reaction products formed by the enzyme. Analysis of the ternary substrates-complex structure and site-directed mutagenesis showed that five residues were involved in the interaction with myo-inositol. Structural comparison with other ribokinase family enzymes indicated that two of the five residues, Q136 and R140, are characteristic of myo-inositol kinase. The crystal structure of the ternary products-complex, which was prepared by incubating the TK2285 protein with myo-inositol and ATP, holds 1d-myo-inositol 3-phosphate (Ins(3)P) in the active site. NMR and HPLC analyses with a chiral column also indicated that the TK2285 reaction product was Ins(3)P. The results obtained here showed that the TK2285 protein specifically catalyzes the phosphorylation of the 3-OH of myo-inositol. We thus designated TK2285 as myo-inositol 3-kinase (MI3K). The precise identification of the reaction product should provide a sound basis to further explore inositol metabolism in Archaea.

Expression, purification, crystallization and X-ray diffraction studies of the molecular chaperone prefoldin from Homo sapiens

Co-reporter:Yoshiki Aikawa;Hiroshi Kida;Yuichi Nishitani

Acta Crystallographica Section F 2015 Volume 71( Issue 9) pp:1189-1193

Publication Date(Web):

DOI:10.1107/S2053230X15013990

Proper protein folding is an essential process for all organisms. Prefoldin (PFD) is a molecular chaperone that assists protein folding by delivering non-native proteins to group II chaperonin. A heterohexamer of eukaryotic PFD has been shown to specifically recognize and deliver non-native actin and tubulin to chaperonin-containing TCP-1 (CCT), but the mechanism of specific recognition is still unclear. To determine its crystal structure, recombinant human PFD was reconstituted, purified and crystallized. X-ray diffraction data were collected to 4.7 Å resolution. The crystals belonged to space group P2₁2₁2, with unit-cell parameters a = 123.2, b = 152.4, c = 105.9 Å.

Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

Co-reporter:Satoshi Watanabe;Takehiko Wada;Kenji Inaba;Haruyuki Atomi;Tamotsu Kanai;Tadayuki Imanaka;Yuichi Nishitani;Takumi Kawashima

PNAS 2015 Volume 112 (Issue 25 ) pp:7701-7706

Publication Date(Web):2015-06-23

DOI:10.1073/pnas.1503102112

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 (HypBAT) with Ni ions. Transient association between HypA and HypBAT is controlled by the ATP hydrolysis cycle of HypBAT, which is accelerated by HypA. Only the ATP-bound form of HypBAT 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 HypABAT complex has a nanomolar affinity (Kd = 7 nM), which is in contrast to the micromolar affinity (Kd = 4 µM) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypBAT, affecting its association with HypA. These findings indicate that HypA and HypBAT constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypBAT functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.

Substrate Distortion Contributes to the Catalysis of Orotidine 5′-Monophosphate Decarboxylase

Co-reporter:Masahiro Fujihashi ; Toyokazu Ishida ; Shingo Kuroda ; Lakshmi P. Kotra ; Emil F. Pai

Journal of the American Chemical Society 2013 Volume 135(Issue 46) pp:17432-17443

Publication Date(Web):October 23, 2013

DOI:10.1021/ja408197k

Orotidine 5′-monophosphate decarboxylase (ODCase) accelerates the decarboxylation of orotidine 5′-monophosphate (OMP) to uridine 5′-monophosphate (UMP) by 17 orders of magnitude. Eight new crystal structures with ligand analogues combined with computational analyses of the enzyme’s short-lived intermediates and the intrinsic electronic energies to distort the substrate and other ligands improve our understanding of the still controversially discussed reaction mechanism. In their respective complexes, 6-methyl-UMP displays significant distortion of its methyl substituent bond, 6-amino-UMP shows the competition between the K72 and C6 substituents for a position close to D70, and the methyl and ethyl esters of OMP both induce rotation of the carboxylate group substituent out of the plane of the pyrimidine ring. Molecular dynamics and quantum mechanics/molecular mechanics computations of the enzyme–substrate complex also show the bond between the carboxylate group and the pyrimidine ring to be distorted, with the distortion contributing a 10–15% decrease of the ΔΔG⧧ value. These results are consistent with ODCase using both substrate distortion and transition-state stabilization, primarily exerted by K72, in its catalysis of the OMP decarboxylation reaction.

Crystal structures of the carbamoylated and cyanated forms of HypE for [NiFe] hydrogenase maturation

Co-reporter:Satoshi Watanabe;Taiga Tominaga;Tadayuki Imanaka;Rie Matsumi;Haruyuki Atomi

PNAS 2013 Volume 110 (Issue 51 ) pp:20485-20490

Publication Date(Web):2013-12-17

DOI:10.1073/pnas.1313620110

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.

Structure Analysis and Comparative Characterization of the Cytochrome c′ and Flavocytochrome c from Thermophilic Purple Photosynthetic Bacterium Thermochromatium tepidum

Co-reporter:Yu Hirano, Yukihiro Kimura, Hideaki Suzuki, Kunio Miki, and Zheng-Yu Wang

Biochemistry 2012 Volume 51(Issue 33) pp:

Publication Date(Web):July 24, 2012

DOI:10.1021/bi3005522

The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c′ and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c′ and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c′ was 22 °C higher than that of Alc. vinosum Cyt c′, and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c′ are substituted by alanines in Tch. tepidum Cyt c′. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c′ and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins.

Crystal structure of the conserved N-terminal domain of the peroxisomal matrix protein import receptor, Pex14p

Co-reporter:Jian-Rong Su;Shigehiko Tamura;Kazuki Takeda;Yukio Fujiki

PNAS 2009 Volume 106 (Issue 2 ) pp:417-421

Publication Date(Web):2009-01-13

DOI:10.1073/pnas.0808681106

Pex14p is a central component of the peroxisomal protein import machinery, in which the conserved N-terminal domain mediates dynamic interactions with other peroxins including Pex5p, Pex13p, and Pex19p. Here, we report the crystal structure of the conserved N-terminal domain of Pex14p with a three-helix bundle. A hydrophobic surface is composed of the conserved residues, of which two phenylalanine residues (Phe-35 and Phe-52) protrude to the solvent. Consequently, two putative binding pockets suitable for recognizing the helical WXXXF/Y motif of Pex5p are formed on the surface by the two phenylalanine residues accompanying with positively charged residues. The structural feature agrees well with our earlier findings where F35A/L36A and F52A/L53A mutants were impaired in the interactions with other peroxins such as Pex5p and Pex13p. Pex14p variants each with Phe-to-Ala mutation at positions 35, 52, and 35/52, respectively, were defective in restoring the impaired peroxisomal protein import in pex14 Chinese hamster ovary mutant ZP161 cells. Moreover, in GST pull-down assays His6-Pex5pL bound only to GST-Pex14p(25–70), not to any of GST-Pex14p(25–70)F35A, GST-Pex14p(25–70)F52A, and GST-Pex14p(25–70)F35A/F52A. Endogenous Pex5p was recruited to FLAG-Pex14p on peroxisomes in vivo but barely to FLAG-Pex14pF35A, FLAG-Pex14pF52A, and FLAG-Pex14pF35A/F52A. Collectively, Phe-35 and Phe-52 are essential for the Pex14p functions, including the interaction between Pex14p and Pex5p.

Structural Basis for the Heterotropic and Homotropic Interactions of Invertebrate Giant Hemoglobin

Co-reporter:Nobutaka Numoto, Taro Nakagawa, Akiko Kita, Yuichi Sasayama, Yoshihiro Fukumori and Kunio Miki

Biochemistry 2008 Volume 47(Issue 43) pp:

Publication Date(Web):October 4, 2008

DOI:10.1021/bi8012609

The oxygen binding properties of extracellular giant hemoglobins (Hbs) in some annelids exhibit features significantly different from those of vertebrate tetrameric Hbs. Annelid giant Hbs show cooperative oxygen binding properties in the presence of inorganic cations, while the cooperativities of vertebrate Hbs are enhanced by small organic anions or chloride ions. To elucidate the structural basis for the cation-mediated cooperative mechanisms of these giant Hbs, we determined the crystal structures of Ca2+- and Mg2+-bound Hbs from Oligobrachia mashikoi at 1.6 and 1.7 Å resolution, respectively. Both of the metal-bound structures were determined in the oxygenated state. Four Ca2+-binding sites and one Mg2+-binding site were identified in each tetramer subassembly. These cations are considered to stabilize the oxygenated form and increase affinity and cooperativity for oxygen binding, as almost all of the Ca2+ and Mg2+ cations were bound at the interface regions, forming either direct or hydrogen bond-mediated interactions with the neighboring subunits. A comparison of the structures of the oxygenated form and the partially unliganded form provides structural insight into proton-coupled cooperativity (Bohr effect) and ligand-induced transitions. Two histidine residues are assumed to be primarily associated with the Bohr effect. With regard to the ligand-induced cooperativity, a novel quaternary rotation mechanism is proposed to exist at the interface region of the dimer subassembly. Interactions among conserved residues Arg E10, His F3, Gln F7, and Val E11, together with the bending motion of the heme molecules, appear to be essential for quaternary rearrangement.

Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA

Co-reporter:Kunio Miki;Akiko Kita;Satoshi Watanabe;Kazuo Kobayashi

PNAS 2008 Volume 105 (Issue 11 ) pp:4121-4126

Publication Date(Web):2008-03-18

DOI:10.1073/pnas.0709188105

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 antioxidant 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 ≈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.

Structural basis for regulation of bifunctional roles in replication initiator protein

Co-reporter:Chieko Wada;Akira Nakamura

PNAS 2007 Volume 104 (Issue 47 ) pp:18484-18489

Publication Date(Web):2007-11-20

DOI:10.1073/pnas.0705623104

DNA replication initiator protein RepE stringently regulates F plasmid replication by its two distinct molecular association states. A predominant dimer functions as an autogenous repressor, whereas monomers act as replication initiators, and the dimer requires actions of the DnaK molecular chaperone system for monomerization. The structure of the monomeric form is known, whereas the dimeric structure and structural details of the dimer-to-monomer conversion have been unclear. Here we present the crystal structure of the RepE dimer in complex with the repE operator DNA. The dimerization interface is mainly formed by intermolecular β-sheets with several key interactions of charged residues. The conformations of the internal N- and C-terminal domains are conserved between the dimer and monomer, whereas the relative domain orientations are strikingly different, allowing for an efficient oligomeric transition of dual-functional RepE. This domain relocation accompanies secondary structural changes in the linker connecting the two domains, and the linker is included in plausible DnaK/DnaJ-binding regions. These findings suggest an activation mechanism for F plasmid replication by RepE monomerization, which is induced and mediated by the DnaK system.

Corrigendum to: “Crystallization and preliminary X-ray crystallographic analysis of extracellular giant hemoglobin from pogonophoran Oligobrachia mashikoi” [Biochim. Biophys. Acta 1750 (2005) 173–176]

Co-reporter:Nobutaka Numoto, Taro Nakagawa, Akiko Kita, Yuichi Sasayama, Yoshihiro Fukumori, Kunio Miki

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2006 Volume 1764(Issue 2) pp:327

Publication Date(Web):February 2006

DOI:10.1016/j.bbapap.2006.01.001

Structure of an extracellular giant hemoglobin of the gutless beard worm Oligobrachia mashikoi

Co-reporter:Nobutaka Numoto;Taro Nakagawa;Akiko Kita;Yuichi Sasayama;Yoshihiro Fukumori

PNAS 2005 102 (41 ) pp:14521-14526

Publication Date(Web):2005-10-11

DOI:10.1073/pnas.0501541102

Mouthless and gutless marine animals, pogonophorans and vestimentiferans, obtain their nutrition solely from their symbiotic chemoautotrophic sulfur-oxidizing bacteria. These animals have sulfide-binding 400-kDa and/or 3,500-kDa Hb, which transports oxygen and sulfide simultaneously. The symbiotic bacteria are supplied with sulfide by Hb of the host animal and use it to provide carbon compounds. Here, we report the crystal structure of a 400-kDa Hb from pogonophoran Oligobrachia mashikoi at 2.85-Å resolution. The structure is hollow-spherical, composed of a total of 24 globins as a dimer of dodecamer. This dodecameric assemblage would be a fundamental structural unit of both 400-kDa and 3,500-kDa Hbs. The structure of the mercury derivative used for phasing provides insights into the sulfide-binding mechanism. The mercury compounds bound to all free Cys residues that have been expected as sulfide-binding sites. Some of the free Cys residues are surrounded by Phe aromatic rings, and mercury atoms come into contact with these residues in the derivative structure. It is strongly suggested that sulfur atoms bound to these sites could be stabilized by aromatic-electrostatic interactions by the surrounding Phe residues.

Crystal structure of thermostable DNA photolyase: Pyrimidine-dimer recognition mechanism

Co-reporter:Hirofumi Komori;Ryoji Masui;Shigeyuki Yokoyama;Seiki Kuramitsu;Takehiko Shibata;Yorinao Inoue

PNAS 2001 Volume 98 (Issue 24 ) pp:13560-13565

Publication Date(Web):2001-11-20

DOI:10.1073/pnas.241371398

DNA photolyase is a pyrimidine-dimer repair enzyme that uses visible light. Photolyase generally contains two chromophore cofactors. One is a catalytic cofactor directly contributing to the repair of a pyrimidine-dimer. The other is a light-harvesting cofactor, which absorbs visible light and transfers energy to the catalytic cofactor. Photolyases are classified according to their second cofactor into either a folate- or deazaflavin-type. The native structures of both types of photolyases have already been determined, but the mechanism of substrate recognition remains largely unclear because of the lack of structural information regarding the photolyase-substrate complex. Photolyase from Thermus thermophilus, the first thermostable class I photolyase found, is favorable for function analysis, but even the type of the second cofactor has not been identified. Here, we report the crystal structures of T. thermophilus photolyase in both forms of the native enzyme and the complex along with a part of its substrate, thymine. A structural comparison with other photolyases suggests that T. thermophilus photolyase has structural features allowing for thermostability and that its light-harvesting cofactor binding site bears a close resemblance to a deazaflavin-type photolyase. One thymine base is found at the hole, a putative substrate-binding site near the catalytic cofactor in the complex form. This structural data for the photolyase-thymine complex allow us to propose a detailed model for the pyrimidine-dimer recognition mechanism.

Crystal structure of cis-prenyl chain elongating enzyme, undecaprenyl diphosphate synthase

Co-reporter:Masahiro Fujihashi;Yuan-Wei Zhang;Yoshiki Higuchi;Xiao-Yuan Li;Tanetoshi Koyama

PNAS 2001 Volume 98 (Issue 8 ) pp:4337-4342

Publication Date(Web):2001-04-10

DOI:10.1073/pnas.071514398

Undecaprenyl diphosphate synthase (UPS) catalyzes the cis-prenyl chain elongation onto trans, trans-farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of bacterial cell walls. We report here the crystal structure of UPS as the only three-dimensional structure among cis-prenyl chain elongating enzymes. The structure is classified into a protein fold family and is completely different from the so-called “isoprenoid synthase fold” that is believed to be a common structure for the enzymes relating to isoprenoid biosynthesis. Conserved amino acid residues among cis-prenyl chain elongating enzymes are located around a large hydrophobic cleft in the UPS structure. A structural P-loop motif, which frequently appears in the various kinds of phosphate binding site, is found at the entrance of this cleft. The catalytic site is determined on the basis of these structural features, from which a possible reaction mechanism is proposed.

Crystal structures of photosynthetic reaction center and high-potential iron-sulfur protein from Thermochromatium tepidum: Thermostability and electron transfer

Co-reporter:Terukazu Nogi;Insan Fathir;Masayuki Kobayashi;Tsunenori Nozawa

PNAS 2000 Volume 97 (Issue 25 ) pp:13561-13566

Publication Date(Web):2000-12-05

DOI:10.1073/pnas.240224997

The reaction center (RC) of photosynthetic bacteria is a membrane protein complex that promotes a light-induced charge separation during the primary process of photosynthesis. In the photosynthetic electron transfer chain, the soluble electron carrier proteins transport electrons to the RC and reduce the photo-oxidized special-pair of bacteriochlorophyll. The high-potential iron-sulfur protein (HiPIP) is known to serve as an electron donor to the RC in some species, where the c-type cytochrome subunit, the peripheral subunit of the RC, directly accepts electrons from the HiPIP. Here we report the crystal structures of the RC and the HiPIP from Thermochromatium (Tch.) tepidum, at 2.2-Å and 1.5-Å resolution, respectively. Tch. tepidum can grow at the highest temperature of all known purple bacteria, and the Tch. tepidum RC shows some degree of stability to high temperature. Comparison with the RCs of mesophiles, such as Blastochloris viridis, has shown that the Tch. tepidum RC possesses more Arg residues at the membrane surface, which might contribute to the stability of this membrane protein. The RC and the HiPIP both possess hydrophobic patches on their respective surfaces, and the HiPIP is expected to interact with the cytochrome subunit by hydrophobic interactions near the heme-1, the most distal heme to the special-pair.

Detailed assessment of X-ray induced structural perturbation in a crystalline state protein

Co-reporter:Kazuki Takeda, Kouji Kusumoto, Yu Hirano, Kunio Miki

Journal of Structural Biology (February 2010) Volume 169(Issue 2) pp:135-144

Publication Date(Web):1 February 2010

DOI:10.1016/j.jsb.2009.09.012

The positions of hydrogen atoms significantly define protein functions. However, such information from protein crystals is easily disturbed by X-rays. The damage can not be prevented completely even in the data collection at cryogenic temperatures. Therefore, the influence of X-rays should be precisely estimated in order to derive meaningful information from the crystallographic results. Diffraction data from a single crystal of the high-potential iron-sulfur protein (HiPIP) from Thermochromatium tepidum were collected at an undulator beamline of a third generation synchrotron facility, and were merged into three data sets according to X-ray dose. A series of structures analyzed at 0.70 Å shows detailed views of the X-ray induced perturbation, such as the positional changes of hydrogen atoms of a water molecule. Based on the results, we successfully collected a low perturbation data set using attenuated X-rays. There was no influence on the crystallographic statistics, such as the relative B factors, during the course of data collection. The electron densities for hydrogen atoms were more clear despite the slightly lower resolution.

Structural and functional characterisation of the cyanobacterial PetC3 Rieske protein family

Co-reporter:Sebastian Veit, Kazuki Takeda, Yuichi Tsunoyama, Frauke Baymann, Reinat Nevo, Ziv Reich, Matthias Rögner, Kunio Miki, Sascha Rexroth

Biochimica et Biophysica Acta (BBA) - Bioenergetics (December 2016) Volume 1857(Issue 12) pp:1879-1891

Publication Date(Web):December 2016

DOI:10.1016/j.bbabio.2016.09.007

Dimer structure and conformational variability in the N-terminal region of an archaeal small heat shock protein, StHsp14.0

Co-reporter:Kazuki Takeda, Takuro Hayashi, Tetsuya Abe, Yu Hirano, Yuya Hanazono, Masafumi Yohda, Kunio Miki

Journal of Structural Biology (April 2011) Volume 174(Issue 1) pp:92-99

Publication Date(Web):1 April 2011

DOI:10.1016/j.jsb.2010.12.006

Small heat shock proteins (sHsps), which are categorized into a class of molecular chaperones, bind and stabilize denatured proteins to prevent aggregation. The sHsps undergo transition between different oligomeric states to control their hydrophobicity. So far, only the structures of sHsps in large oligomeric states have been reported. Here we report the structure of StHsp14.0 from Sulfolobus tokodaii in the dimeric state, which is formed by means of a mutation at the C-terminal IXI/V motif. The dimer is the sole building block in two crystal forms, and the dimeric mode is the same as that in the large oligomers. The N-terminal helix has variety in its conformation. Furthermore, spectroscopic and biochemical experiments were performed to investigate the conformational variability at the N-terminus. The structural, dynamical and oligomeric properties suggest that chaperone activity of StHsp14.0 is mediated by partially dissolved oligomers.

Structure Analysis of Archaeal AMP Phosphorylase Reveals Two Unique Modes of Dimerization

Co-reporter:Yuichi Nishitani, Riku Aono, Akira Nakamura, Takaaki Sato, ... Kunio Miki

Journal of Molecular Biology (9 August 2013) Volume 425(Issue 15) pp:2709-2721

Publication Date(Web):9 August 2013

DOI:10.1016/j.jmb.2013.04.026

•AMPpase is an essential enzyme in an archaeal AMP metabolic pathway.•Tk-AMPpase eluted in the void volume of a gel-filtration column.•Two distinct interfaces for dimerization are important for the multimerization.•The N-terminal domain also participates in both multimerization and domain closure.•AMPpase is distinct in function from previously known members of the nucleoside phosphorylase II family.AMP phosphorylase (AMPpase) catalyzes the initial reaction in a novel AMP metabolic pathway recently found in archaea, converting AMP and phosphate into adenine and ribose 1,5-bisphosphate. Gel-filtration chromatography revealed that AMPpase from Thermococcus kodakarensis (Tk-AMPpase) forms an exceptionally large macromolecular structure (> 40-mers) in solution. To investigate its unique multimerization feature, we determined the first crystal structures of Tk-AMPpase, in the apo-form and in complex with substrates. Structures of two truncated forms of Tk-AMPpase (Tk-AMPpaseΔN84 and Tk-AMPpaseΔC10) clarified that this multimerization is achieved by two dimer interfaces within a single molecule: one by the central domain and the other by the C-terminal domain, which consists of an unexpected domain-swapping interaction. The N-terminal domain, characteristic of archaeal enzymes, is essential for enzymatic activity, participating in multimerization as well as domain closure of the active site upon substrate binding. Moreover, biochemical analysis demonstrated that the macromolecular assembly of Tk-AMPpase contributes to its high thermostability, essential for an enzyme from a hyperthermophile. Our findings unveil a unique archaeal nucleotide phosphorylase that is distinct in both function and structure from previously known members of the nucleoside phosphorylase II family.Download high-res image (359KB)Download full-size image

Structural Studies on the Oligomeric Transition of a Small Heat Shock Protein, StHsp14.0

Co-reporter:Yuya Hanazono, Kazuki Takeda, Masafumi Yohda, Kunio Miki

Journal of Molecular Biology (7 September 2012) Volume 422(Issue 1) pp:100-108

Publication Date(Web):7 September 2012

DOI:10.1016/j.jmb.2012.05.017

The small heat shock proteins (sHsps), which are widely found in all domains of life, bind and stabilize denatured proteins to prevent aggregation. The sHsps exist as large oligomers that are composed of 9–40 subunits and control their chaperone activity by the transition of the oligomeric state. Though the oligomeric transition is important for the biological function of most sHsps, atomic details have not been elucidated. Here, we report crystal structures in both the 24-meric and dimeric states for an sHsp, StHsp14.0 from Sulfolobus tokodaii, in order to reveal changes upon the oligomeric transition. The results indicate that StHsp14.0 forms a spherical 24-mer with a diameter of 115 Å. The diameter is defined by the inter-monomer angle in the dimer. The dimer structure in the dimeric state shows only small differences from that in the 24-meric state. Some significant differences are exclusively observed at the binding site for the C-terminus. Although a dimer has four interactive sites with neighboring dimers, the weakness of the respective interactions is indicated from the size-exclusion chromatography. The small structural changes imply an activation mechanism mediated by multiple weak interactions.Download high-res image (306KB)Download full-size imageResearch Highlights► Crystal structures in the 24-meric and dimeric states of StHsp14.0 were determined. ► Structural differences are exclusively observed at the binding site for C-terminus. ► The dimer structure is ready for the formation of the 24-mer. ► The respective interactions between the IXI/V motif and the binding site are very weak. ► The small structural changes imply an activation mediated by weak interactions.

Crystal Structures of [NiFe] Hydrogenase Maturation Proteins HypC, HypD, and HypE: Insights into Cyanation Reaction by Thiol Redox Signaling

Co-reporter:Satoshi Watanabe, Rie Matsumi, Takayuki Arai, Haruyuki Atomi, ... Kunio Miki

Molecular Cell (6 July 2007) Volume 27(Issue 1) pp:29-40

Publication Date(Web):6 July 2007

DOI:10.1016/j.molcel.2007.05.039

[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 Å, 2.07 Å, and 1.55 Å 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.

Crystal Structure of a Thermophilic GrpE Protein: Insight into Thermosensing Function for the DnaK Chaperone System

Co-reporter:Akira Nakamura, Kouhei Takumi, Kunio Miki

Journal of Molecular Biology (5 March 2010) Volume 396(Issue 4) pp:1000-1011

Publication Date(Web):5 March 2010

DOI:10.1016/j.jmb.2009.12.028

A homodimeric GrpE protein functions as a nucleotide exchange factor of the eubacterium DnaK molecular chaperone system. The co-chaperone GrpE accelerates ADP dissociation from, and promotes ATP binding to, DnaK, which cooperatively facilitates the DnaK chaperone cycle with another co-chaperone, DnaJ. GrpE characteristically undergoes two-step conformational changes in response to elevation of the environmental temperature. In the first transition at heat-shock temperatures, a fully reversible and functionally deficient structural alteration takes place in GrpE, and then the higher temperatures lead to the irreversible dissociation of the GrpE dimer into monomers as the second transition. GrpE is also thought to be a thermosensor of the DnaK system, since it is the only member of the DnaK system that changes its structure reversibly and loses its function at heat-shock temperatures of various organisms. We here report the crystal structure of GrpE from Thermus thermophilus HB8 (GrpETth) at 3.23 Å resolution. The resolved structure is compared with that of GrpE from mesophilic Escherichia coli (GrpEEco), revealing structural similarities, particularly in the DnaK interaction regions, and structural characteristics for the thermal stability of GrpETth. In addition, the structure analysis raised the possibility that the polypeptide chain in the reported GrpEEco structure was misinterpreted. Comparison of these two GrpE structures combined with the results of limited proteolysis experiments provides insight into the protein dynamics of GrpETth correlated with the shift of temperature, and also suggests that the localized and partial unfolding at the plausible DnaK interaction sites of GrpETth causes functional deficiency of nucleotide exchange factor in response to the heat shock.

Nonequivalence Observed for the 16-Meric Structure of a Small Heat Shock Protein, SpHsp16.0, from Schizosaccharomyces pombe

Co-reporter:Yuya Hanazono, Kazuki Takeda, Toshihiko Oka, Tetsuya Abe, ... Kunio Miki

Structure (5 February 2013) Volume 21(Issue 2) pp:220-228

Publication Date(Web):5 February 2013

DOI:10.1016/j.str.2012.11.015

Small heat shock proteins (sHsps) play a role in preventing the fatal aggregation of denatured proteins in the presence of stresses. The sHsps exist as monodisperse oligomers in their resting state. Because the hydrophobic N-terminal regions of sHsps are possible interaction sites for denatured proteins, the manner of assembly of the oligomer is critical for the activation and inactivation mechanisms. Here, we report the oligomer architecture of SpHsp16.0 from Schizosaccharomyces pombe determined with X-ray crystallography and small angle X-ray scattering. Both results indicate that eight dimers of SpHsp16.0 form an elongated sphere with 422 symmetry. The monomers show nonequivalence in the interaction with neighboring monomers and conformations of the N- and C-terminal regions. Variants for the N-terminal phenylalanine residues indicate that the oligomer formation ability is highly correlated with chaperone activity. Structural and biophysical results are discussed in terms of their possible relevance to the activation mechanism of SpHsp16.0.Highlights► Eight dimers of SpHsp16.0 form an elongated sphere with 422 symmetry ► SAXS model is well fitted into the crystal structure ► Monomers show nonequivalence in the interaction and conformations ► Oligomer formation ability of variants is highly correlated with chaperone activity

Crystal Structures of the HypCD Complex and the HypCDE Ternary Complex: Transient Intermediate Complexes during [NiFe] Hydrogenase Maturation

Co-reporter:Satoshi Watanabe, Rie Matsumi, Haruyuki Atomi, Tadayuki Imanaka, Kunio Miki

Structure (5 December 2012) Volume 20(Issue 12) pp:2124-2137

Publication Date(Web):5 December 2012

DOI:10.1016/j.str.2012.09.018

[NiFe] hydrogenase maturation represents one of the most dynamic and sophisticated processes in metallocenter assembly. The Fe(CN)2CO 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.Highlights► Crystal structures of the HypCD and HypCDE ternary complexes were determined ► Conserved hydrophobic residues mainly form the HypC-HypD interface ► The HypCDE complex formation renders the HypE conformation suitable for CN transfer ► The conserved motifs of the three proteins are assembled in the ternary complex

Crystal Structure of HypA, a Nickel-Binding Metallochaperone for [NiFe] Hydrogenase Maturation

Co-reporter:Satoshi Watanabe, Takayuki Arai, Rie Matsumi, Haruyuki Atomi, ... Kunio Miki

Journal of Molecular Biology (4 December 2009) Volume 394(Issue 3) pp:448-459

Publication Date(Web):4 December 2009

DOI:10.1016/j.jmb.2009.09.030

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.

Crystal Structure of the Stationary Phase Survival Protein SurE with Metal Ion and AMP

Co-reporter:Wakana Iwasaki, Kunio Miki

Journal of Molecular Biology (3 August 2007) Volume 371(Issue 1) pp:123-136

Publication Date(Web):3 August 2007

DOI:10.1016/j.jmb.2007.05.007

The stationary phase survival protein SurE is a metal ion-dependent phosphatase distributed among eubacteria, archaea, and eukaryotes. In Escherichia coli, SurE has activities as nucleotidase and exopolyphosphatase, and is thought to be involved in stress response. However, its physiological role and reaction mechanism are unclear. We report here the crystal structures of the tetramer of SurE from Thermus thermophilus HB8 (TtSurE) both alone and crystallized with Mn2+ and substrate AMP. In the presence of Mn2+ and AMP, differences between the protomers were observed in the active site and in the loop located near the active site; AMP-bound active sites with the loops in a novel open conformation were found in the two protomers, and AMP-free active sites with the loops in a conventional closed conformation were found in the other two protomers. The two loops in the open conformation are entwined with each other, and this entwining is suggested to be required for enzymatic activity by site-directed mutagenesis. TtSurE exists as an equilibrium mixture of dimer and tetramer in solution. The loop-entwined structure indicates that SurE acts as a tetramer. The structural features and the absence of negative cooperativity imply the half-of-the-sites reactivity mechanism resulting from a pre-existing tendency toward structural asymmetry.

Crystal Structure of Archaeal Photolyase from Sulfolobus tokodaii with Two FAD Molecules: Implication of a Novel Light-harvesting Cofactor

Co-reporter:Masahiro Fujihashi, Nobutaka Numoto, Yukiko Kobayashi, Akira Mizushima, ... Kunio Miki

Journal of Molecular Biology (26 January 2007) Volume 365(Issue 4) pp:903-910

Publication Date(Web):26 January 2007

DOI:10.1016/j.jmb.2006.10.012

UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.

Crystal Structure of a Periplasmic Substrate-Binding Protein in Complex with Calcium Lactate

Co-reporter:Nobuhiko Akiyama, Kazuki Takeda, Kunio Miki

Journal of Molecular Biology (25 September 2009) Volume 392(Issue 3) pp:559-565

Publication Date(Web):25 September 2009

DOI:10.1016/j.jmb.2009.07.043

Lactate is utilized in many biological processes, and its transport across biological membranes is mediated with various types of transporters. Here, we report the crystal structures of a lactate-binding protein of a TRAP (tripartite ATP-independent periplasmic) secondary transporter from Thermus thermophilus HB8. The folding of the protein is typical for a type II periplasmic solute-binding protein and forms a dimer in a back-to-back manner. One molecule of l-lactate is clearly identified in a cleft of the protein as a complex with a calcium ion. Detailed crystallographic and biochemical analyses revealed that the calcium ion can be removed from the protein and replaced with other divalent cations. This characterization of the structure of a protein binding with calcium lactate makes a significant contribution to our understanding of the mechanisms by which calcium and lactate are accommodated in cells.

Structure and Mutation Analysis of Archaeal Geranylgeranyl Reductase

Co-reporter:Daisuke Sasaki, Masahiro Fujihashi, Yuki Iwata, Motomichi Murakami, ... Kunio Miki

Journal of Molecular Biology (17 June 2011) Volume 409(Issue 4) pp:543-557

Publication Date(Web):17 June 2011

DOI:10.1016/j.jmb.2011.04.002

The crystal structure of geranylgeranyl reductase (GGR) from Sulfolobus acidocaldarius was determined in order to elucidate the molecular mechanism of the catalytic reaction. The enzyme is a flavoprotein and is involved in saturation of the double bonds on the isoprenoid moiety of archaeal membranes. The structure determined in this study belongs to the p-hydroxybenzoate hydroxylase family in the glutathione reductase superfamily. GGR functions as a monomer and is divided into the FAD-binding, catalytic and C-terminal domains. The catalytic domain has a large cavity surrounded by a characteristic YxWxFPx7-8GxG motif and by the isoalloxazine ring of an FAD molecule. The cavity holds a lipid molecule, which is probably derived from Escherichia coli cells used for over-expression. One of the two forms of the structure clarifies the presence of an anion pocket holding a pyrophosphate molecule, which might anchor the phosphate head of the natural ligands. Mutational analysis supports the suggestion that the three aromatic residues of the YxWxFPx7-8GxG motif hold the ligand in the appropriate position for reduction. Cys47, which is widely conserved in GGRs, is located at the si-side of the isoalloxazine ring of FAD and is shown by mutational analysis to be involved in catalysis. The catalytic cycle, including the FAD reducing factor binding site, is proposed on the basis of the detailed analysis of the structure.Download high-res image (137KB)Download full-size imageResearch Highlights► The crystal structure of an archaeal geranylgeranyl reductase was determined. ► A lipid molecule is bound at the ligand-binding cavity next to FAD. ► The structure and mutation analysis identify the residues important for catalysis.

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

Co-reporter:Yu Hirano, Makoto Higuchi, Chihiro Azai, Hirozo Oh-oka, ... Zheng-Yu Wang

Journal of Molecular Biology (16 April 2010) Volume 397(Issue 5) pp:1175-1187

Publication Date(Web):16 April 2010

DOI:10.1016/j.jmb.2010.02.011

In green sulfur photosynthetic bacteria, the cytochrome cz (cyt cz) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt cz subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt cz subunit (C-cyt cz) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt cz consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt cz supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt cz.

Elucidations of the Catalytic Cycle of NADH-Cytochrome b5 Reductase by X-ray Crystallography: New Insights into Regulation of Efficient Electron Transfer

Co-reporter:Mitsugu Yamada, Taro Tamada, Kazuki Takeda, Fumiko Matsumoto, ... Kunio Miki

Journal of Molecular Biology (15 November 2013) Volume 425(Issue 22) pp:4295-4306

Publication Date(Web):15 November 2013

DOI:10.1016/j.jmb.2013.06.010

•The crystal structure of the fully reduced b5R was determined at 1.68 Å resolution.•In the reduced form, domain motion and a flat FAD isoalloxazine ring were observed.•The oxidized form denotes the highest resolution ever achieved for a flavoprotein.•The final step of the catalytic cycle was confirmed by the cryo-trapping method.NADH-Cytochrome b5 reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b5 (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68 Å resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD+. Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78 Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.Download high-res image (323KB)Download full-size image

Structural and Molecular Characterization of the Prefoldin β Subunit from Thermococcus Strain KS-1

Co-reporter:Hiroshi Kida, Yuri Sugano, Ryo Iizuka, Masahiro Fujihashi, ... Kunio Miki

Journal of Molecular Biology (14 November 2008) Volume 383(Issue 3) pp:465-474

Publication Date(Web):14 November 2008

DOI:10.1016/j.jmb.2008.08.041

Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of α and β subunits and forms a “jellyfish-like” structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two α and two β subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of α (α1 and α2) and β subunits (β1 and β2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD β1 subunit at 1.9 Å resolution and its functional analysis. TsPFD β1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The β hairpin linkers of β1 subunits assemble to form a β barrel “body” around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the β1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric β1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD β1 subunits act as molecular chaperones in living cells of some archaea.

Structural Studies of the Cpx Pathway Activator NlpE on the Outer Membrane of Escherichia coli

Co-reporter:Yu Hirano, Md. Motarab Hossain, Kazuki Takeda, Hajime Tokuda, Kunio Miki

Structure (14 August 2007) Volume 15(Issue 8) pp:963-976

Publication Date(Web):14 August 2007

DOI:10.1016/j.str.2007.06.014

NlpE, an outer membrane lipoprotein, functions during envelope stress responses in Gram-negative bacteria. In Escherichia coli, adhesion to abiotic surfaces has been reported to activate the Cpx pathway in an NlpE-dependent manner. External copper ions are also thought to activate the Cpx pathway mediated by NlpE. We determined the crystal structure of NlpE from E. coli at 2.6 Å resolution. The structure showed that NlpE consists of two β barrel domains. The N-terminal domain resembles the bacterial lipocalin Blc, and the C-terminal domain has an oligonucleotide/oligosaccharide-binding (OB) fold. From both biochemical analyses and the crystal structure, it can be deduced that the cysteine residues in the CXXC motif may be chemically active. Furthermore, two monomers in the asymmetric unit form an unusual 3D domain-swapped dimer. These findings indicate that tertiary and/or quaternary structural instability may be responsible for Cpx pathway activation.