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Daisuke Kohda Last modified date:2023.06.05

Graduate School
Administration Post

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Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University .
Structural Life Science, Medical Life Sciences, Graduate School of Systems Life Sciences .
Academic Degree
Doctor of Science
Country of degree conferring institution (Overseas)
Field of Specialization
Structural Biology, Biochemistry
ORCID(Open Researcher and Contributor ID)
Total Priod of education and research career in the foreign country
Outline Activities
Structural biology is a special field of biology concerned with the molecular structure of biological macromolecules and the molecular basis of their functions. Our structural biology is characterized as follows: we use an ingenious combination of X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and single particle analysis and tomography using cryoelectron microscopy. These methods are complemented by expertise in the fields of protein expression and purification, and analyses of protein-ligand interactions with various physicochemical techniques. Our target biological systems include (1) Mitochondrial protein import system; (2) Oligosaccharyltransferase that catalyzes glycosylation on asparagine residues in protein; (3) Proteins involved in DNA replication, repair, and recombination; (4) Proteins involved in clathrin-mediated endocytosis.
It is common knowledge that a protein binds to its target ligand specifically based on the lock-and-key mechanism, but we are very interested in molecular recognitions with promiscuous specificity (i.e., nonstrict specificity with weak affinity). A novel mechanism could be involved, and we think that it should be a “molecular frustration”. New techniques are required to study a novel mechanism. We are developing or using new experimental techniques to solve the so-called sampling problem in structure determination: ▶ Shift the dynamic equilibrium of protein-ligand interactions to the bound state, by molecular tethering and molecular stiffening techniques. ▶ Creation of “contact-effect-free space” in protein crystals. We are also developing a new NMR experiment to reveal the physicochemical basis of fast and smooth protein folding. ▶ The linear free energy relationship between log K and log k on a per-residue basis.
In sum, we are promoting structure determinations of protein-protein complexes and supramolecular protein complexes using these new techniques under the concept of “structure at work”.
Research Interests
  • Physicochemical basis of protein folding
    keyword : NMR, thermodynamic analysis, kinetic analysis
  • NMR dynamic study of the lantibiotic peptide, Nukacin ISK-1
    keyword : lantibiotics, NMR, thermodynamic analysis, kinetic analysis
  • SAXS measurement of distance distribution function between two specific atoms in proteins
    keyword : SAXS, protein, heavy atom labeling, distance distribution function
  • Creation of crystal contact-free space in protein crystals for the study of protein dynamics
    keyword : X-ray crystallography, protein, crystallization, dynamics
  • Crystallographic study of proteins involved in chloroplast photorelocation
    keyword : chloroplast photorelocation
  • Comparative structural biology of oligosaccharyltransferases from various organisms
    keyword : oligosaccharytransferase, N-glycosylation
    2003.01Comparative structural biology of oligosaccharyltransferases from various organisms.
  • Structural Biology of Tom20 and Tom22 proteins involved in protein transport into the mitochondrial matrix
    keyword : mitochondria, protein import, presequence
    1998.01Structural Biology of Tom20 and Tom40 proteins involved in protein transport into the mitochondrial matrix.
  • Structural Biology of protein domains
    keyword : protein domains, SH3, PX, etc
    1992.01~2008.03Structural Biology of protein domains: SH3, PX, LINK,PXA.
  • Structural basis of the 3' terminal recognition of DNA by the PriA protein
    keyword : PriA, 3' terminus of DNA, stalled DNA replication fork
    2000.01~2008.03Structural basis of the 3' terminal recognition of DNA by the PriA protein.
Current and Past Project
  • Protein 3000 project
Academic Activities
1. Hidekazu Hiroaki, Daisuke Kohda, Protein-ligand interactions studied by NMR, Springer Singapore, 10.1007/978-981-10-5966-7_21, 579-600, 2017.11, Various solution NMR experiments for studying protein-ligand interactions have become indispensable techniques in both academia and industry. In general, solution NMR is superior to other physico-chemical methods, in terms of its spatial resolution and the fact that protein modifications are not required. The applications are loosely classified into two categories, "ligand-based approach" and "protein-based approach." Many unique experiments have been developed for the ligand-based approach, including STD, WaterLOGSY, DIRECTION, INPHARMA, ILOE, and trNOE. These experiments frequently comprise the important steps of a drug-discovery process, including ligand screening, pharmacophore mapping, and molecular design. This review provides a practicable classification of these experiments, to promote the selection of a suitable experiment depending on the purpose. In contrast, the variation of experiments in the protein-based approach is rather limited. The 1H-15N-HSQC-based NMR titration experiment and its variants are preferentially used for analyses of protein-ligand interactions. This review also discusses several practical aspects of the NMR titration experiment, including sample handling and data acquisition and analysis..
1. Fujinami D, Hayashi S, Kohda D, Retrospective study for the universal applicability of the residue-based linear free energy relationship in the two-state exchange of protein molecules, Scientific Report, 10.1038/s41598-022-21226-z, 12, 1, 16843, 2022.10.
2. Han X, Maita N, Shimada A, Kohda D, Effects of targeting signal mutations in a mitochondrial presequence on the spatial distribution of the conformational ensemble in the binding site of Tom20, Protein Science, 10.1002/pro.4433, 31, 10, 4433, 2022.10.
3. Hayashi S, Kohda D, The time-zero HSQC method improves the linear free energy relationship of a polypeptide chain through the accurate measurement of residue-specific equilibrium constants, J Biomolecular NMR, 10.1007/s10858-022-00396-y, 76, 3, 87-94, 2022.06.
4. Fujinami D, Hayashi S, Kohda D., Residue-Specific Kinetic Insights into the Transition State in Slow Polypeptide Topological Isomerization by NMR Exchange Spectroscopy, J Phys Chem Lett, 10.1021/acs.jpclett.1c02387, 12, 43, 10551-10557, 2021.11.
5. 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..
6. 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..
7. 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..
8. 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..
9. 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.
10. 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.
11. 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.
12. 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.
13. 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.
14. 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.
15. 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..
16. 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.
17. 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.
1. Daisuke Kohda, The lantibiotic nukacin ISK-1 exists in an equilibrium between active and inactive lipid-II binding states: Thermodynamic analysis using the NMR signals as residue-specific probes, Annual Taiwan Magnetic Resonance Society Meeting, 2019.12.
2. Daisuke Kohda, The lantibiotic nukacin ISK-1 exists in an equilibrium between active and inactive lipid-II binding states , 8th Asia-Pacific NMR Symposium 2019, 2019.07.
3. Daisuke Kohda, Integrative Structural Biology Approach to Understand the Structural and Dynamic Basis of Asn-Glycosylation, The Japanese Biochemical Society Bio-Frontier Symposium, International symposium on ER stress, glycosylation, homeostasis and diseases, 2018.03.
4. Daisuke Kohda, Integrative structural biology approach to decipher the molecular mechanism of Asn-glycosylation , The 42nd Naito Conference, “In the Vanguard of Structural Biology: Revolutionizing Life Sciences”, 2016.10.
5. Daisuke Kohda, Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation , 2014 SFG & JSCR joint annual meeting, 2014.11.
6. Daisuke Kohda, Crystal structures of oligosaccharyltransferase provide insights into the sequon recognition and activation of N-linked protein glycosylation, 第87回日本生化学会大会シンポジウム, 2014.10.
7. Daisuke Kohda, Structural and dynamic basis for mitochondrial presequence recognition by Tom20, ISN 2014 Special Neurochemistry Conference, 2014.09.
8. Daisuke Kohda, Intentional creation of crystal-contact free space for analyzing large amplitude motions in protein crystals , International Symposium between Kyushu U. Post Global COE and School of Biomedical Sciences, Monash U., 2014.02.
9. 神田 大輔, Intentional creation of crystal-contact free space for monitoring large amplitude motions of proteins and ligands in protein crystals, 第85回日本生化学会年会, 2012.12.
10. 神田 大輔, Structural biology of the N-glycosylation reaction, The 20th Anniversary of the Mizutani Foundation for Glycosciences, 2012.11.
11. Cracking of the targeting signal embedded in mitochondrial presequences by NMR and crystallography
Daisuke Kohda
CREST international symposium, Frontier of Biologcal NMR Spectroscopy.
12. From structure to function of PX domains in the NADPH oxidase system
Daisuke Kohda
11th Hot Spring Harbor Symposium of Medical Institute of Bioregulation.
13. Structure and Function of PX and SH3 Domains in the NAPDH Oxidase System
Daisuke Kohda
the 2nd Tsinghua International Conference of Protein Science
Tsinghua University, Beijing, China.
Membership in Academic Society
  • Japan Consortium for Glycobiology and Glycotechnology
  • the Crystallographic Society of Japan
  • The Japanese Society of Carbohydrate Research
  • the Nuclear Magnetic Resonance Society of Japan
  • the Biophysical Society of Japan
  • the Molecular Biology Society of Japan
  • the Protein Science Society of Japan
  • the Japanese Biochemical Society
Educational Activities
A part of regular teaching in medical life science I and Topics in medical life science III provided by systems life sciences, and in other courses provided by graduate school of medicine and faculty of arts and science of Kyushu University.
Other educational activities out of Kyushu University is as follows:
July 2004, lecture, The University of Tokyo
February 2005, lecture, KAST course
June 2005, lecture, The University of Tokyo
July 2008, lecture, Kumamoto University
December 2008, open lecture in the 46th annual meeting of the Biophysical Society of Japan
June 2009, lecture, Hokkaido University
November 2010, lecture, Yokohama City University
July 2012, lecture in the 13th NMR Wakate Kenkyukai (Hokkaido)
May 2018, lecture, Osaka University
October 2018, lecture, Kobe University
June 2019, lecture on integrated structural biology, Kobe
November 2019, lecture on integrated structural biology, Tokyo
Other Educational Activities
  • 2020.03.
  • 2019.11.
  • 2018.11.
  • 2017.01.
  • 2013.09.
  • 2011.09.
  • 2012.10.
  • 2010.09.