Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Daisuke Kohda Last modified date:2021.10.23

Professor / Division of Structural Biology / Department of Molecular and Structural Biology / Medical Institute of Bioregulation

1. Yuya Taguchi, Takahiro Yamasaki, Marie Ishikawa, Yuki Kawasaki, Ryuji Yukimura, Maki Mitani, Kunio Hirata, Daisuke Kohda, The structure of an archaeal oligosaccharyltransferase provides insight into the strict exclusion of proline from the N-glycosylation sequon, Communication Biology, 10.1038/s42003-021-02473-8, 4, 1, 941, 2021.08, Oligosaccharyltransferase (OST) catalyzes oligosaccharide transfer to the Asn residue in the N-glycosylation sequon, Asn-X-Ser/Thr, where Pro is strictly excluded at position X. Considering the unique structural properties of proline, this exclusion may not be surprising, but the structural basis for the rejection of Pro residues should be explained explicitly. Here we determined the crystal structure of an archaeal OST in a complex with a sequon-containing peptide and dolichol-phosphate to a 2.7 Å resolution. The sequon part in the peptide forms two inter-chain hydrogen bonds with a conserved amino acid motif, TIXE. We confirmed the essential role of the TIXE motif and the adjacent regions by extensive alanine-scanning of the external loop 5. A Ramachandran plot revealed that the ring structure of the Pro side chain is incompatible with the ϕ backbone dihedral angle around -150° in the rigid sequon-TIXE structure. The present structure clearly provides the structural basis for the exclusion of Pro residues from the N-glycosylation sequon..
2. Naomi Inoue, Takeshi Terabayashi, Yuri Takiguchi-Kawashima, Daisuke Fujinami, Shigeru Matsuoka, Masanori Kawano, Kazuhiro Tanaka, Hiroshi Tsumura, Toshimasa Ishizaki, Hisashi Narahara, Daisuke Kohda, Yoshihiro Nishida, Katsuhiro Hanada, The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants, Scientific Report, 10.1038/s41598-021-87344-2, 11, 1, 7718, 2021.04, DNA replication inhibitors are utilized extensively in studies of molecular biology and as chemotherapy agents in clinical settings. The inhibition of DNA replication often triggers double-stranded DNA breaks (DSBs) at stalled DNA replication sites, resulting in cytotoxicity. In East Asia, some traditional medicines are administered as anticancer drugs, although the mechanisms underlying their pharmacological effects are not entirely understood. In this study, we screened Japanese herbal medicines and identified two benzylisoquinoline alkaloids (BIAs), berberine and coptisine. These alkaloids mildly induced DSBs, and this effect was dependent on the function of topoisomerase I (Topo I) and MUS81-EME1 structure-specific endonuclease. Biochemical analysis revealed that the action of BIAs involves inhibiting the catalytic activity of Topo I rather than inducing the accumulation of the Topo I-DNA complex, which is different from the action of camptothecin (CPT). Furthermore, the results showed that BIAs can act as inhibitors of Topo I, even against CPT-resistant mutants, and that the action of these BIAs was independent of CPT. These results suggest that using a combination of BIAs and CPT might increase their efficiency in eliminating cancer cells..
3. Kouta Mayanagi, Keisuke Oki, Naoyuki Miyazaki, Sonoko Ishino, Takeshi Yamagami, Kosuke Morikawa, Kenji Iwasaki, Daisuke Kohda, Tsuyoshi Shirai, Yoshizumi Ishino, Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy, BMC Biology, 10.1186/s12915-020-00889-y, 18, 1, 152, 2020.10, Background: DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3'-5' editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM).

Results: Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a β-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro.

Conclusions: Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life..
4. Yuki Kawasaki, Hirotaka Ariyama, Hajime Motomura, Daisuke Fujinami, Daisuke Noshiro, Toshio Ando, Daisuke Kohda, Two-State Exchange Dynamics in Membrane-Embedded Oligosaccharyltransferase Observed in Real-Time by High-Speed AFM, Journal of Molecular Biology, 10.1016/j.jmb.2020.09.017, 432, 22, 5951-5965, 2020.11, Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of oligosaccharide chains from lipid-linked oligosaccharides (LLO) to asparagine residues in polypeptide chains. Using high-speed atomic force microscopy (AFM), we investigated the dynamic properties of OST molecules embedded in biomembranes. An archaeal single-subunit OST protein was immobilized on a mica support via biotin-avidin interactions and reconstituted in a lipid bilayer. The distance between the top of the protein molecule and the upper surface of the lipid bilayer was monitored in real-time. The height of the extramembranous part exhibited a two-step variation with a difference of 1.8 nm. The high and low states are designated as state 1 and state 2, respectively. The transition processes between the two states fit well to single exponential functions, suggesting that the observed dynamic exchange is an intrinsic property of the archaeal OST protein. The two sets of cross peaks in the NMR spectra of the protein supported the conformational changes between the two states in detergent-solubilized conditions. Considering the height values measured in the AFM measurements, state 1 is closer to the crystal structure, and state 2 has a more compact form. Subsequent AFM experiments indicated that the binding of the sugar donor LLO decreased the structural fluctuation and shifted the equilibrium almost completely to state 1. This dynamic behavior is likely necessary for efficient catalytic turnover. Presumably, state 2 facilitates the immediate release of the bulky glycosylated polypeptide product, thus allowing OST to quickly prepare for the next catalytic cycle..
5. Yuya Taguchi, Tomohide Saio, Daisuke Kohda, Distance Distribution between Two Iodine Atoms Derived from Small-Angle X-ray Scattering Interferometry for Analyzing a Conformational Ensemble of Heavy Atom-Labeled Small Molecules, Journal of Physical Chemistry Letters, 10.1021/acs.jpclett.0c01107, 11, 14, 5451-5456, 2020.07, To obtain unbiased information about the dynamic conformational ensemble of a molecule in solution, one promising approach is small-angle X-ray scattering (SAXS). Conventionally, SAXS data are converted to a pair distribution function, which describes the distance distribution between all pairs of atoms within a molecule. If two strong X-ray scatterers are introduced and the background contributions from the other atoms are suppressed, then the distance distribution between the two scatterers provides spatial information about a flexible molecule. Gold nanocrystals can provide such information for distances of >50 Å. Here, we synthesized a chemical compound containing two iodine atoms attached to the ends of a flexible polyethylene glycol chain and used the relevant singly labeled and unlabeled compounds to suppress the background contribution. This is a feasibility demonstration to prove that the distance distribution in the range of 10-30 Å can be experimentally accessed by SAXS..
6. Takahiro Yamasaki, Daisuke Kohda, Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase, The Journal of biological chemistry, 10.1074/jbc.RA120.015013, 2020.09, Oligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST hydrolyzes the oligosaccharide donor, releasing free N-glycans (FNGs) into the lumen of the endoplasmic reticulum (ER). Here, we established a purification method for mutated OSTs using a high-affinity epitope tag attached to the catalytic subunit Stt3, from yeast cells co-expressing the wild-type OST to support growth. The purified OST protein with mutations is useful for wide-ranging biochemical experiments. We assessed the effects of mutations in the Stt3 subunit on the two enzymatic activities in vitro, as well as their effects on the N-glycan attachment and FNG content levels in yeast cells. We found that mutations in the first DXD motif increased the FNG generation activity relative to the oligosaccharyl transfer activity, both in vitro and in vivo, while mutations in the DK motif had the opposite effect; the decoupling of the two activities may facilitate future deconvolution of the reaction mechanism. The isolation of the mutated OSTs also enabled us to identify different enzymatic properties in OST complexes containing either the Ost3 or Ost6 subunit and to find a 15-residue peptide as a better quality substrate than shorter peptides. This toolbox of mutants, substrates, and methods will be useful for investigations of the molecular basis and physiological roles of the OST enzymes in yeast and other organisms..
7. Ryo Akiyama, Masahiko Annaka, Daisuke Kohda, Hiroyuki Kubota, Yusuke Maeda, Nobuaki Matsumori, Daisuke Mizuno, Norio Yoshida, Biophysics at Kyushu University, Biophysical Reviews, 10.1007/s12551-020-00643-2, 12, 2, 245-247, 2020.04.
8. Daisuke Fujinami, Hajime Motomura, Hiraku Oshima, Abdullah Al Mahin, Khaled M. Elsayed, Takeshi Zendo, Yuji Sugita, Kenji Sonomoto, Daisuke Kohda, Mosaic Cooperativity in Slow Polypeptide Topological Isomerization Revealed by Residue-Specific NMR Thermodynamic Analysis, Journal of Physical Chemistry Letters, 10.1021/acs.jpclett.9b03591, 11, 5, 1934-1939, 2020.03, Slow polypeptide conformational changes on time scales of >1 s are generally assumed to be highly cooperative two-state transitions, reflecting the high energy barrier. However, few experimental characterizations have tested the validity of this assumption. We performed residue-specific NMR thermodynamic analysis of the 27-residue lantibiotic peptide, nukacin ISK-1, to characterize the isomerization between two topological states on the second time scale. Unexpectedly, the thermal transition behaviors were distinct among peptide regions, indicating that the topological isomerization process is a mosaic of different degrees of cooperativity. The conformational change path between the two NMR structures was deduced by a targeted molecular dynamics simulation. The unique side-chain threading motions through the monosulfide rings are the structural basis of the high energy barrier, and the nonlocal interactions in the hydrophobic core are the structural basis of the cooperativity. Taken together, we provide an energetic description of the topological isomerization of nukacin ISK-1..
9. Kyoko Hanawa-Suetsugu, Yuzuru Itoh, Maisarah Ab Fatah, Tamako Nishimura, Kazuhiro Takemura, Kohei Takeshita, Satoru Kubota, Naoyuki Miyazaki, Wan Nurul Izzati Wan Mohamad Noor, Takehiko Inaba, Nhung Thi Hong Nguyen, Sayaka Hamada-Nakahara, Kayoko Oono-Yakura, Masashi Tachikawa, Kenji Iwasaki, Daisuke Kohda, Masaki Yamamoto, Akio Kitao, Atsushi Shimada, Shiro Suetsugu, Phagocytosis is mediated by two-dimensional assemblies of the F-BAR protein GAS7, Nature communications, 10.1038/s41467-019-12738-w, 10, 1, 2019.12, Phagocytosis is a cellular process for internalization of micron-sized large particles including pathogens. The Bin-Amphiphysin-Rvs167 (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, impose specific morphologies on lipid membranes. Most BAR domain proteins are thought to form membrane invaginations or protrusions by assembling into helical submicron-diameter filaments, such as on clathrin-coated pits, caveolae, and filopodia. However, the mechanism by which BAR domain proteins assemble into micron-scale phagocytic cups was unclear. Here, we show that the two-dimensional sheet-like assembly of Growth Arrest-Specific 7 (GAS7) plays a critical role in phagocytic cup formation in macrophages. GAS7 has the F-BAR domain that possesses unique hydrophilic loops for two-dimensional sheet formation on flat membranes. Super-resolution microscopy reveals the similar assemblies of GAS7 on phagocytic cups and liposomes. The mutations of the loops abolishes both the membrane localization of GAS7 and phagocytosis. Thus, the sheet-like assembly of GAS7 plays a significant role in phagocytosis..
10. Takahiro Yamasaki, Daisuke Kohda, A Radioisotope-free Oligosaccharyltransferase Assay Method., Bio-protocol, 10.21769/BioProtoc.3186, 9, 5, 2019.03, [URL], Glycosylation of asparagine residues is widespread in Eukarya, and occurs in virtually all Archaea and some eubacterial species. A membrane-bound enzyme, oligosaccharyltransferase, catalyzes the transfer of an oligosaccharide chain from a sugar donor (lipid-linked oligosaccharide, LLO) to an asparagine residue in the consensus sequence, Asn-X-Ser/Thr (X ≠ Pro), in proteins. The in vitro oligosaccharyl transfer assay reaction mixture contains a detergent-solubilized oligosaccharyltransferase (OST), a sugar donor LLO, and a sugar acceptor peptide. Previous assay methods are problematic, in terms of the use of radioactive compounds and the cumbersome separation procedures using lectin binding or two-phase partitioning. Here, we describe a new oligosaccharyl transfer assay method, which is radioisotope-free and relies on a different separation mechanism. The glycopeptide products are separated from unreacted peptides by SDS-PAGE. A fluorescent dye is attached to the peptide substrate during custom peptide synthesis. The fluorescent imaging of the SDS-PAGE gels ensures high sensitivity and quantitative performance. The user-friendly PAGE format is particularly suitable for presentation in scientific papers. For illustrative applications, time-course and peptide library experiments are shown..
11. Arpita Srivastava, Florence Tama, Daisuke Kohda, Osamu Miyashita, Computational investigation of the conformational dynamics in Tom20-mitochondrial presequence tethered complexes, Proteins: Structure, Function and Bioinformatics, 10.1002/prot.25625, 87, 1, 81-90, 2019.01, The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes..
12. Arpita Srivastava, Siqin Bala, Hajime Motomura, Daisuke Kohda, Florence Tama, Osamu Miyashita, Conformational ensemble of an intrinsically flexible loop in mitochondrial import protein Tim21 studied by modeling and molecular dynamics simulations, Biochimica et Biophysica Acta - General Subjects, 10.1016/j.bbagen.2019.129417, 1864, 2, 2019.01, Background: Tim21, a subunit of a highly dynamic translocase of the inner mitochondrial membrane (TIM23) complex, translocates proteins by interacting with subunits in the translocase of the outer membrane (TOM) complex and Tim23 channel in the TIM23 complex. A loop segment in Tim21, which is in close proximity of the binding site of Tim23, has different conformations in X-ray, NMR and new crystal contact-free space (CCFS) structures. MD simulations can provide information on the structure and dynamics of the loop in solution. Methods: The conformational ensemble of the loop was characterized using loop modeling and molecular dynamics (MD) simulations. Results: MD simulations confirmed mobility of the loop. Multidimensional scaling and clustering were used to characterize the dynamic conformational ensemble of the loop. Free energy landscape showed that the CCFS crystal structure occupied a low energy region as compared to the conventional X-ray crystal structure. Analysis of crystal packing indicates that the CCFS provides larger conformational space for the motions of the loop. Conclusions: Our work reported the conformational ensemble of the loop in solution, which is in agreement with the structure obtained from CCFS approach. The combination of the experimental techniques and computational methods is beneficial for studying highly flexible regions of proteins. General significance: Computational methods, such as loop modeling and MD simulations, have proved to be useful for studying conformational flexibility of proteins. These methods in integration with experimental techniques such as CCFS has the potential to transform the studies on flexible regions of proteins..
13. Siqin Bala, Shoko Shinya, Arpita Srivastava, Marie Ishikawa, Atsushi Shimada, Naohiro Kobayashi, Chojiro Kojima, Florence Tama, Osamu Miyashita, Daisuke Kohda, Crystal contact-free conformation of an intrinsically flexible loop in protein crystal
Tim21 as the case study, Biochimica et Biophysica Acta - General Subjects, 10.1016/j.bbagen.2019.129418, 1864, 2, 2019.01, Background: In protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules. Methods: We designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal. Results: Yeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures. Conclusions: Loop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution. General significance: No single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal..
14. Noritaka Nishida, Tomoya Tsukazaki, Daisuke Kohda, Preface to the special issue on novel measurement techniques for visualizing ‘live’ protein molecules at work, Biochimica et Biophysica Acta - General Subjects, 10.1016/j.bbagen.2019.129421, 1864, 2, 2019.01.
15. Kouta Mayanagi, Sonoko Ishino, Tsuyoshi Shirai, Takuji Oyama, Shinichi Kiyonari, Daisuke Kohda, Kosuke Morikawa, Yoshizumi Ishino, Direct visualization of DNA baton pass between replication factors bound to PCNA, Scientific reports, 10.1038/s41598-018-34176-2, 8, 1, 2018.12, In Eukarya and Archaea, the lagging strand synthesis is accomplished mainly by three key factors, DNA polymerase (Pol), flap endonuclease (FEN), and DNA ligase (Lig), in the DNA replication process. These three factors form important complexes with proliferating cell nuclear antigen (PCNA), thereby constructing a platform that enable each protein factor to act successively and smoothly on DNA. The structures of the Pol-PCNA-DNA and Lig-PCNA-DNA complexes alone have been visualized by single particle analysis. However, the FEN-PCNA-DNA complex structure remains unknown. In this report, we for the first time present this tertiary structure determined by single particle analysis. We also successfully visualized the structure of the FEN-Lig-PCNA-DNA complex, corresponding to a putative intermediate state between the removal of the DNA flap by FEN and the sealing of the nicked DNA by Lig. This structural study presents the direct visualization of the handing-over action, which proceeds between different replication factors on a single PCNA clamp bound to DNA. We detected a drastic conversion of the DNA from a bent form to a straight form, in addition to the dynamic motions of replication factors in the switching process..
16. Daisuke Fujinami, -Mahin Abdullah Al, Khaled M. Elsayed, Mohammad R. Islam, Jun ichi Nagao, Urmi Roy, Sabrina Momin, Takeshi Zendo, Daisuke Kohda, Kenji Sonomoto, The lantibiotic nukacin ISK-1 exists in an equilibrium between active and inactive lipid-II binding states, Communications Biology, 10.1038/s42003-018-0150-3, 1, 1, 2018.12, The lantibiotic nukacin ISK-1 exerts antimicrobial activity through binding to lipid II. Here, we perform NMR analyses of the structure of nukacin ISK-1 and the interaction with lipid II. Unexpectedly, nukacin ISK-1 exists in two structural states in aqueous solution, with an interconversion rate on a time scale of seconds. The two structures differ in the relative orientations of the two lanthionine rings, ring A and ring C. Chemical shift perturbation induced by the titration of lipid II reveals that only one state was capable of binding to lipid II. On the molecular surface of the active state, a multiple hydrogen-bonding site formed by amino acid residues in the ring A region is adjacent to a hydrophobic surface formed by residues in the ring C region, and we propose that these sites interact with the pyrophosphate moiety and the isoprene chain of the lipid II molecule, respectively..
17. Daisuke Kohda, “Multiple partial recognitions in dynamic equilibrium” in the binding sites of proteins form the molecular basis of promiscuous recognition of structurally diverse ligands, Biophysical Reviews, 10.1007/s12551-017-0365-4, 10, 2, 421-433, 2018.04, Promiscuous recognition of ligands by proteins is as important as strict recognition in numerous biological processes. In living cells, many short, linear amino acid motifs function as targeting signals in proteins to specify the final destination of the protein transport. In general, the target signal is defined by a consensus sequence containing wild-characters, and hence represented by diverse amino acid sequences. The classical lock-and-key or induced-fit/conformational selection mechanism may not cover all aspects of the promiscuous recognition. On the basis of our crystallographic and NMR studies on the mitochondrial Tom20 protein–presequence interaction, we proposed a new hypothetical mechanism based on “a rapid equilibrium of multiple states with partial recognitions”. This dynamic, multiple recognition mode enables the Tom20 receptor to recognize diverse mitochondrial presequences with nearly equal affinities. The plant Tom20 is evolutionally unrelated to the animal Tom20 in our study, but is a functional homolog of the animal/fungal Tom20. NMR studies by another research group revealed that the presequence binding by the plant Tom20 was not fully explained by simple interaction modes, suggesting the presence of a similar dynamic, multiple recognition mode. Circumstantial evidence also suggested that similar dynamic mechanisms may be applicable to other promiscuous recognitions of signal peptides by the SRP54/Ffh and SecA proteins..
18. Yuya Taguchi, Daisuke Fujinami, Daisuke Kohda, Comparative analysis of the oligosaccharide donors (Lipid-linked oligosaccharides) for the n-oligosaccharyl transfer reaction, Trends in Glycoscience and Glycotechnology, 10.4052/tigg.1721.4E, 30, 176, 2018.01, The glycosylation on asparagine residues is one of the ubiquitous protein modifications occurring in the three domains of life. An oligosaccharide chain is preassembled on a lipid-phospho carrier, which is referred to as lipid-linked oligosaccharide (LLO), and transferred to asparagine residues in polypeptide chains by the action of a membrane-bound enzyme, oligosaccharyltransferase. The oligosaccharide donor for the oligosaccharyl transfer reaction is dolichol–diphosphate–oligosaccharide in Eukarya, and polyprenol–diphosphate–oligosaccharide in Eubacteria. The oligosaccharide donor of an archaeal species was reported as dolichol–monophosphate–oligosaccharide. Thus, the difference in the number of phosphate groups aroused interest in whether the use of the monophosphate type donor is widespread in the domain Archaea. In this study, we selected four archaeal species to widely sample the domain Archaea. We used normal-phase liquid chromatography to purify the LLO from cultured archaeal cells and performed ESI-MS analysis to determine the chemical structure of the lipid-phospho part. We found that two euryarchaeal oligosaccharide donors of more ancient origin were a dolichol-monophosphate type, whereas the two crenarchaeal oligosaccharide donors of closer origin to Eukarya were a dolichol–diphosphate type. The present comparative study provides a new insight into the evolution of the oligosaccharide donor in the N-glycosylation system..
19. Daisuke Kohda, Structural basis of protein asn-glycosylation by oligosaccharyltransferases, Advances in Experimental Medicine and Biology, 10.1007/978-981-13-2158-0_9, 171-199, 2018.01, Glycosylation of asparagine residues is a ubiquitous protein modification. This N-glycosylation is essential in Eukaryotes, but principally nonessential in Prokaryotes (Archaea and Eubacteria), although it facilitates their survival and pathogenicity. In many reviews, Archaea have received far less attention than Eubacteria, but this review will cover the N-glycosylation in the three domains of life. The oligosaccharide chain is preassembled on a lipid-phospho carrier to form a donor substrate, lipid-linked oligosaccharide (LLO). The en bloc transfer of an oligosaccharide from LLO to selected Asn residues in the Asn-X-Ser/Thr (X≠Pro) sequons in a polypeptide chain is catalyzed by a membrane-bound enzyme, oligosaccharyltransferase (OST). Over the last 10 years, the three-dimensional structures of the catalytic subunits of the Stt3/AglB/PglB proteins, with an acceptor peptide and a donor LLO, have been determined by X-ray crystallography, and recently the complex structures with other subunits have been determined by cryo-electron microscopy. Structural comparisons within the same species and across the different domains of life yielded a unified view of the structures and functions of OSTs. A catalytic structure in the TM region accounts for the amide bond twisting, which increases the reactivity of the side-chain nitrogen atom of the acceptor Asn residue in the sequon. The Ser/Thr-binding pocket in the C-terminal domain explains the requirement for hydroxy amino acid residues in the sequon. As expected, the two functional structures are formed by the involvement of short amino acid motifs conserved across the three domains of life..
20. Daisuke Fujinami, Yuya Taguchi, Daisuke Kohda, Asn-linked oligosaccharide chain of a crenarchaeon, Pyrobaculum calidifontis, is reminiscent of the eukaryotic high-mannose-type glycan., Glycobiology, 10.1093/glycob/cwx044, 2017.05.
21. Sunsuke Matsumoto, Yuya Taguchi, Atsushi Shimada, Mayumi Igura, Daisuke Kohda, Tethering an N-Glycosylation Sequon-Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex., Biochemistry, 10.1021/acs.biochem.6b01089, 56, 4, 602-611, 2017.01.
22. Rei Matsuoka, Atsushi Shimada, Yasuaki Komuro, Yuji Sugita, Daisuke Kohda, Rational design of crystal contact-free space in protein crystals for analyzing spatial distribution of motions within protein molecules, Protein Science, 10.1002/pro.2867, 25, 3, 754-768, 2016.03.
23. Yuya Taguchi, Daisuke Fujinami, Daisuke Kohda, Comparative analysis of archaeal lipid-linked oligosaccharides that serve as oligosaccharide donors for Asn-glycosylation, Journal Biological Chemistry, 2016.03.
24. Atsushi Shimada, Atsuko Yamaguchi, Daisuke Kohda, Structural basis for the recognition of two consecutive mutually interacting DPF motifs by the SGIP1 μ homology domain, Scientific Report, 10.1038/srep19565, 29, 6, 19565, 2016.01.
25. Daisuke Fujinami, James Nyirenda, Shunsuke Matsumoto, Daisuke Kohda, Structural elucidation of an asparagine-linked oligosaccharide from the hyperthermophilic archaeon, Archaeoglobus fulgidus, CARBOHYDRATE RESEARCH, 10.1016/j.carres.2015.05.010, 413, 55-62, 2015.06.
26. Daisuke Fujinami, Masaki Matsumoto, Takuya Noguchi, Kenji Sonomoto, Daisuke Kohda, Structural elucidation of an asparagine-linked oligosaccharide from the hyperthermophilic archaeon, Pyrococcus furiosus, CARBOHYDRATE RESEARCH, 10.1016/j.carres.2014.01.021, 387, 30-36, 2014.03.
27. 松本 俊介, Atsushi Shimada, James Nyirenda, 井倉 真由美, Yoshiaki Kawano, Daisuke Kohda, Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1309777110, 110, 44, 17868-17873, 2013.10.
28. 松本 俊介, Atsushi Shimada, Daisuke Kohda, Crystal structure of the C-terminal globular domain of the third paralog of the Archaeoglobus fulgidus oligosaccharyltransferases, BMC STRUCTURAL BIOLOGY, 10.1186/1472-6807-13-11, 13, 11, 2013.07.
29. Komuro, Y., Miyashita, N., Mori, T., Muneyuki, E., Takashi Saitoh, Daisuke Kohda, Sugita, Y., Energetics of the Presequence-Binding Poses in Mitochondrial Protein Import Through Tom20, J. Phys. Chem. Part B, 117, 2864-2871, 2013.03.
30. Nyirenda, J., 松本 俊介, Takashi Saitoh, Maita, N.,, Noda, N. N., Inagaki, F., Daisuke Kohda, Crystallographic and NMR evidence for flexibility in oligosaccharyltransferases and its catalytic significance, Structure, 21, 32-41, 2013.01.
31. 松本 俊介, 井倉 真由美, Nyirenda, J., Masaki Matsumoto, Satoru Yuzawa, Noda, N., Inagaki, F., Daisuke Kohda, Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 Å resolution, Biochemistry, 51, 4157-4166, 2012.05.
32. Saitoh T, Igura M, Miyazaki Y, Ose T, Maita N, Kohda D, Crystallographic snapshots of tom20-mitochondrial presequence interactions with disulfide-stabilized peptides., Biochemistry, 50, 24, 5487-5496, 2011.06.
33. Igura M, Kohda D, Quantitative assessment of the preferences for the amino acid residues flanking archaeal N-linked glycosylation sites, Glycobiology, 21, 5, 575-583, 2011.05.
34. Igura M, Kohda D, Selective control of oligosaccharide transfer efficiency for the N-glycosylation sequon by a point mutation in oligosaccharyltransferase , J Biol Chem, 286, 15, 13255-13260, 2011.04, アスパラギン残基の糖鎖修飾は蛋白質の翻訳後修飾のなかで最大のものであり,真核生物,古細菌および一部の真正細菌に普遍的にみられる.オリゴ糖転移酵素は脂質結合型の糖鎖を蛋白質のアスパラギン残基へ転移する反応を触媒する.N型糖鎖修飾のコンセンサス配列であるAsn-X-Ser/Thrにおいて,古細菌Pyrococcus fuirosusではXの位置の最適のアミノ酸はバリンである.古細菌酵素のシステマティックなアラニン置換実験を行い,酵素活性に必須なアミノ酸残基と必須ではないアミノ酸残基を同定した.このうち,必須ではないアミノ酸残基について,その置換がXの位置における酵素活性の好みに与える影響を調べた.すると,一つの位置で影響を与えることがわかった.この残基は保存モチーフであるDXXKXXX(M/I) motifの中のKの位置であった.たとえば,Kを他のアミノ酸に置換することで,Xがアルギニンのコンセンス配列に対し,ワイルドタイプの酵素に比べて9倍もの高い活性を示すことがわかった..
35. Yamamoto H, Itoh N, Kawano S, Yatsukawa Y, Momose T, Makio T, Matsunaga M, Yokota M, Esaki M, Shodai T, Kohda D, Hobbs AE, Jensen RE, Endo T, Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria. , Proc Natl Acad Sci U S A, 108, 1, 91-96, 2011.01.
36. Suetsugu N, Takano A, Kohda D, Structure and activity of JAC1 J-domain implicate the involvement of the cochaperone activity with HSC70 in chloroplast photorelocation movement., Plant Signal Behav, 5, 12, 1602-1606, 2010.12.
37. Masai H, Tanaka T, Kohda D, Stalled replication forks: making ends meet for recognition and stabilization. , Bioessays, 32, 8, 687-697, 2010.08.
38. Takano A, Suetsugu N, Wada M, Kohda D, Crystallographic and functional analyses of J-domain of JAC1 essential for chloroplast photorelocation movement in Arabidopsis thaliana., Plant Cell Physiol, 51, 8, 1372-1376, 2010.08.
39. Maita N, Nyirenda J, Igura M, Kamishikiryo J, Kohda D, Comparative structural biology of eubacterial and archaeal oligosaccharyltransferases., J. Biol. Chem. , 285, 4941-4950, 2010.02.
40. Ichikawa S, Takai T, Yashiki T, Takahashi S, Okumura K, Ogawa H, Kohda D, Hatanaka H., Lipopolysaccharide binding of the mite allergen Der f 2., Genes to Cells, 14, 1055-1065, 2009.08.
41. M. Fukao, T. Obita, F. Yoneyama, D. Kohda, T. Zendo, J. Nakayama, K. Sonomoto, Complete Covalent Structure of Nisin Q, New Natural Nisin Variant, Containing Post-Translationally Modified Amino Acids., Biosci Biotechnol Biochem, 72, 1750-1755, 2008.06.
42. Hashiguchi T, Kajikawa M, Maita N, Takeda M, Kuroki K, Sasaki K, Kohda D, Yanagi Y, Maenaka K., Homogeneous sugar modification improves crystallization of measles virus hemagglutinin, J Virol Methods, 149, 171-174, 2008.04.
43. N. Aoki, A. Sakiyama, K. Kuroki, K. Maenaka, D. Kohda, M. Deshimaru, S. Terada, Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum., Biochim Biophys Acta, 1784, 621-628, 2008.04.
44. S. Tabata, K. Kuroki, J. Wang, M. Kajikawa, I. Shiratori, D. Kohda, H. Arase, K. Maenaka, Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors., J Biol Chem , 283, 8893-8901, 2008.04.
45. S. Tabata, K. Kuroki, N. Maita, J. Wang, I. Shiratori, H. Arase, D. Kohda, K. Maenaka, Expression, crystallization and preliminary X-ray diffraction analysis of human paired Ig-like type 2 receptor alpha (PILRalpha), Acta Crystallogr Sect F Struct Biol Cryst Commun, 64, 44-46, 2008.01.
46. Igura M, Maita N, Kamishikiryo J, Yamada M, Obita T, Maenaka K, Kohda D., Structure-guided identification of a new catalytic motif of oligosaccharyltransferase, EMBO J, 27, 234-243, 2008.01.
47. T. Hashiguchi, M. Kajikawa, N. Maita, M. Takeda, K. Kuroki, K. Sasaki, D. Kohda, Y. Yanagi, K. Maenaka, Crystal structure of measles virus hemagglutinin provides insight into effective vaccines, Proc Natl Acad Sci U S A , 104, 19535-19540, 2007.12.
48. D. Kohda, M. Yamada, M. Igura, J. Kamishikiryo, K. Maenaka, New oligosaccharyltransferase assay method, Glycobiology , 17, 1175-1182, 2007.11.
49. T. Saitoh, M. Igura, T. Obita, T. Ose, R. Kojima, K. Maenaka, T. Endo, D. Kohda, Tom20 recognizes mitochondrial presequences through dynamic equilibrium among multiple bound states, EMBO J , 26, 4777-4787, 2007.11.
50. M. Igura, N. Maita, T. Obita, J. Kamishikiryo, K. Maenaka, D. Kohda, Purification, crystallization and preliminary X-ray diffraction studies of the soluble domain of the oligosaccharyltransferase STT3 subunit from the thermophilic archaeon Pyrococcus furiosus, Acta Crystallogr Sect F Struct Biol Cryst Commun, 63, 798-801, 2007.09.
51. T. Tanaka, T. Mizukoshi, K. Sasaki, D. Kohda, H. Masai, Escherichia coli PriA protein, two modes of DNA binding and activation of ATP hydrolysis, J Biol Chem , 282, 19917-19927, 2007.06.
52. T. Ose, N. Soler, L. Rasubala, K. Kuroki, D. Kohda, D. Fourmy, S. Yoshizawa, K. Maenaka, Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB, Structure, 15, 577-586, 2007.05.
53. K. Kuroki, S. Kobayashi, M. Shiroishi, M. Kajikawa, N. Okamoto, D. Kohda, K. Maenaka, Detection of weak ligand interactions of leukocyte Ig-like receptor B1 by fluorescence correlation spectroscopy, J. Immunol. Methods, 2007.01.
54. T. Ose, N. Soler, L. Rasubala, K. Kuroki, D. Kohda, D. Fourmy, S. Yoshizawa, K. Maenaka, Structural basis for dynamical interdomain movement and RNA recognition of the selenocysteine specific elongation factor SelB, Structure, 2007.01.
55. K. Sasaki, T. Ose, N. Okamoto, K. Maenaka, T. Tanaka, H. Masai, M. Saito, T. Shirai, D. Kohda, Structural basis of the 3'-end recognition of a leading strand in stalled replication forks by PriA, EMBO J., 2007.01.
56. Sasaki, K., Ose, T., Tanaka, T., Mizukoshi, T., Ishigaki, T., Maenaka, K., Masai, H., and Kohda, D., Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli, Biochim Biophys Acta, 1764, 157-160, 2006.01.
57. Shiroishi, M., Kajikawa, M., Kuroki, K., Ose, T., Kohda, D., and Maenaka, K., Crystal structure of the human monocyte-activating receptor, "Group 2" leukocyte Ig-like receptor A5 (LILRA5/LIR9/ILT11)., J Biol Chem, 281, 19536-19544, 2006.01.
58. Shiroishi, M., Kohda, D., and Maenaka, K., Preparation and crystallization of the disulfide-linked HLA-G dimer., Biochim Biophys Acta, 1764, 985-988, 2006.01.
59. Shiroishi, M., Kuroki, K., Ose, T., Rasubala, L., Shiratori, I., Arase, H., Tsumoto, K., Kumagai, I., Kohda, D., and Maenaka, K., Efficient leukocyte Ig-like receptor signaling and crystal structure of disulfide-linked HLA-G dimer., J Biol Chem, 281, 10439-10447, 2006.01.
60. Shiroishi, M., Kuroki, K., Tsumoto, K., Yokota, A., Sasaki, T., Amano, K., Shimojima, T., Shirakihara, Y., Rasubala, L., van der Merwe, P.A., Kumagai, I., Kohda, D., and Maenaka, K., Entropically driven MHC class I recognition by human inhibitory receptor leukocyte Ig-like receptor B1 (LILRB1/ILT2/CD85j)., J Mol Biol, 355, 237-248, 2006.01.
61. I. Nobuhisa, R. Takeya, K. Ogura, N. Ueno, D. Kohda, F. Inagaki, H. Sumimoto, Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent alpha-helix of p22phox, Biochem. J., 396, 183-192, 2006.01.
62. M. Shiroishi, K. Kuroki, L. Rasubala, K. Tsumoto, I. Kumagai, E. Kurimoto, K. Kato, D. Kohda, K. Maenaka, Structural basis for recognition of the nonclassical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d), Proc. Natl. Acad. Sci. U S A. , 103, 16412-16417, 2006.01.
63. S. Shioi, T. Ose, K. Maenaka, M. Shiroishi, Y. Abe, D. Kohda, T. Katayama, T. Ueda, Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site, Biochem. Biophys. Res. Commun, 10.1016/j.bbrc.2004.11.104, 326, 4, 766-776, 326, 766-776, 2005.01.
64. S. Yoshizawa, L. Rasubala, T. Ose, D. Kohda, D. Fourmy, K. Maenaka, Structural basis for mRNA recognition by elongation factor SelB, Nat. Struct. Mol. Biol, 10.1038/nsmb890, 12, 2, 198-203, 12, 198-203, 2005.01.
65. L. Rasubala, D. Fourmy, T. Ose, D. Kohda, K. Maenaka, S. Yoshizawa, Crystallization and preliminary C-ray analysis of the mRNA-binding domain of elongation factor SelB in complex with RNA, Acta Cryst., 10.1107/S1744309105003611, 61, 296-298, F61, 296-298, 2005.01.
66. M. Igura, T. Ose, T. Obita, C. Sato, K. Maenaka, T. Endo, D. Kohda, Crystallization and preliminary X-ray analysis of mitochondrial presequence receptor Tom20 in complexes with a presequence from aldehyde dehydrogenase., Acta Cryst, 10.1107/S1744309105011577, 61, 514-517, F61, 514-517, 2005.01.
67. T. Kamura, K. Maenaka, S. Kotoshiba, M. Matsumoto, D. Kohda, R. C. Conaway, J. W. Conaway, and K. Nakayama, VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases, Genes Dev, 10.1101/gad.1252404, 18, 24, 3055-3065, 18, 3055-3065, 2004.01.
68. T. Obita, T. Muto, T. Endo, and D. Kohda, Peptide library approach with a disulfide tether to refine the Tom20 recognition motif in mitochondrial presequences, J. Mol. Biol., 328, 495-504, 2003.01.
69. T. Ago, F. Kuribayashi, H. Hiroaki, R. Takeya, T. Ito, D. Kohda, H. Sumimoto., Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation., Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0735712100, 100, 8, 4474-4479, 100, 4474-4479, 2003.01.
70. M. Shimizu, D. Kohda, E. H., Morita, S. Hotta, K. Morikawa, H. Hiroaki, 1H, 13C and 15N backbone resonance assignments of the N-terminal domain of Drosophila GCM protein, J. Biomol. NMR, 26, 277-278, 2003.01.
71. M. Shimizu, H. Hiroaki, D. Kohda, T. Hosoya, Y. Akiyama-Oda, Y. Hotta, E. H. Morita, and K. Morikawa, NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+-binding motif, Protein Eng., 10.1093/proeng/gzg040, 16, 4, 247-254, 16, 247-254, 2003.01.
72. Mizukoshi, T., Tanaka, T., Arai, K., Kohda, D., H. Masai, A critical role of the 3ユ-terminus of nascent DNA chains in recognition of stalled replication forks, J. Biol. Chem., 278, 42234-42239, 2003.01.
73. H. Urata, H. Shimizu, H. Hiroaki, D. Kohda, M. Akagi, Thermodynamic study of hybridization properties of heterochiral nucleic acids, Biochem Biophys Res Commun., 10.1016/S0006-291X(03)01531-6, 309, 1, 79-83, 309, 79-83, 2003.01.
74. K. Kami, K., R. Takeya., H. Sumimoto, D. Kohda, Diverse recognition of non-PxxP peptide ligands by the SH3 domains from p67phox, Grb2 and Pex13p, EMBO J., 10.1093/emboj/cdf428, 21, 16, 4268-4276, 21, 4268-4276, 2002.01.
75. T. Endo, D. Kohda, Functions of Outer Membrane Receptors in Mitochondrial Protein Import, Biochim. Biophys. Acta, 10.1016/S0167-4889(02)00259-8, 1592, 1, 3-14, 1592, 3-14, 2002.01.
76. T. Tanaka, T. Mizukoshi, C. Taniyama, D. Kohda, K. Arai, H. Masai, DNA binding of PriA protein requires cooperation of the N-terminal Dloop/arrested-fork binding and C-terminal helicase domains, J. Biol. Chem., 277, 38062-38071, 2002.01.
77. T. Muto, T. Obita, Y. Abe, T. Shodai, T. Endo, D. Kohda, NMR Identification of the Tom20 Binding Element in Mitochondiral Presequences, J. Mol. Biol., 306, 137-143, 2001.01.
78. A. A. Bocquier, L. Liu, I. K. Cann, K. Komori, D. Kohda, Y. Ishino, Archaeal Primase. Bridging the Gap between RNA and DNA Polymerases, Curr. Biol., 11, 452-456, 2001.01.
79. H. Hiroaki, T. Ago, T. Ito, H. Sumimoto, D. Kohda, Solution Structure of the PX domain, a Target of the SH3 domain., Nature Struct. Biol., 10.1038/88591, 8, 6, 526-530, 8, 526-530, 2001.01.
80. T. Ago, R. Takeya, H. Hiroaki, F. Kuribayashi, T. Ito, D. Kohda, H. Sumimoto, The PX Domain as a Novel Phosphoinositide-Binding Module, Biochem. Biophys. Res. Commun., 10.1006/bbrc.2001.5629, 287, 3, 733-738, 287, 733-738, 2001.01.
81. L. Liu, K. Komori, S. Ishino, A. A. Bocquier, I. K. Cann, D. Kohda, Y. Ishino, The archaeal DNA primase: biochemical characterization of the p41-p46 complex from Pyrococcus furiosus, J. Biol. Chem., 276, 45484-45490, 2001.01.
82. A. Kashiwada, H. Hiroaki, D. Kohda, M. Nango, and T. Tanaka, Design of a Heterotrimetric _-Helical Bundle by Hydrophobic Core Engineering, J. Am. Chem. Soc., 122, 212-215, 2000.01.
83. C. Sato, J.-H. Kim, Y. Abe, K. Saito, S. Yokoyama, D. Kohda, Characterization of the N-oligosaccharides attached to the atypical Asn-X-Cys sequence of recombinant human epidermal growth factor receptor, J. Biochem (Tokyo), 127, 65-72, 2000.01.
84. Y. Abe, Toshihiro S., T. Muto, K. Mihara, H. Torii, S. Nishikawa, T. Endo, and D. Kohda, Structural Basis of Presequence Recognition by the Mitochondrial Protein Import Receptor Tom20, Cell, 10.1016/S0092-8674(00)80691-1, 100, 5, 551-560, 100, 551-560, 2000.01.
85. X. Li, K. Suzuki, K. Kanaori, K. Tajima, A. Kashiwada, H. Hiroaki, D. Kohda, T. Tanaka, Soft Metal Ions, Cd(II) and Hg(II), Induce Triple-Stranded _-Helical Assembly and Folding of a de nove Designed Peptide in Their Trigonal Geometries, Protein Sci., 9, 1327-1333, 2000.01.
86. S. E. Tsutakawa, T. Muto, T. Kawate, H. Jingami, N. Kunishima, M. Ariyoshi, D. Kohda, M. Nakagawa, K. Morikawa, Crystallographic and Functional Studies of Very Short Patch Repair Endonuclease, Mol. Cell, 3, 621-628, 1999.01.
87. Y. Abe, M. Odaka, F. Inagaki, I. Lax, J. Schlessinger, D. Kohda, Disulfide Bond Structure of Human Epidermal Growth Factor Receptor, J. Biol. Chem., 273, 11150-11157, 1998.01.
88. K. Suzuki, H. Hiroaki, D. Kohda, T. Tanaka, An Isoleucine Zipper Peptide Forms a Native-like Triple Stranded Coiled Coil in Solution, Protein Engineering, 11, 1051-1055, 1998.01.
89. K. Suzuki, H. Hiroaki, D. Kohda, H. Nakamura, T. Tanaka, Metal Ion Induced Self-Assembly of a Designed Peptide into a Triple-Stranded _-Helical Bundle: A Novel Metal Binding Site in the Hydrophobic Core, J. Am. Chem. Soc., 120, 13008-13015, 1998.01.