Division of Biomolecular and Structural Biology
Main Research Areas
Our main focus is detemination of macromolecular structure of biologically important proteins using X-ray crystallography (Fig.1). Especially our interest is protein-protein complex and protein-DNA complex. Latest research area was described under. We are collaborating with various biochemistry laboratories. We wish to provide important discovery based on crystal structures　of biological macromolecule.

(1) Papain-CLIK148 complex
Cathepsin L is one of the most powerful lysozomal cysteine protease to degrade many proteins and has been implicated as participating in bone collagen degradation by osteoclasts. Cathpsin L specific inhibitor CLIK148 was developed by Prof.Katunuma. We detemined the crystal structure of papain-CLIK148 complex and revealed the binding mode (Fig. 2). The similarity and differece can be seen in the binding between CLIK148 and ZPACK.

(2) Crystal structure of Clostridium perfringens Ia
Iota-toxin from Clostridium perfringens type E is ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals. It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. We reported on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 Å and 2.1 Å resolution, respectively) and the mutagenesis that map the active residues. The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region. However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT.

(3) Crystal structure of ADP-dependent glucokinases from P. horikoshii Although ATP is the most common phosphoryl group donor for kinases, some kinases from certain hyperthermophilic archaea such as Pyrococcus horikoshii and Thermococcus litoralis utilize ADP as the phosphoryl donor. Those are ADP-dependent glucokinases (ADPGK) and phosphofructokinases (ADPPFK) in their glycolytic pathway.
Here, we succeeded in gene cloning the ADPGK from P. horikoshii OT3 (phGK) in Escherichia coli, easy preparation of the enzyme, crystallization and the structure determination of the apo enzyme. We observed a large conformational change between the apo-phGK and ADP-tlGK structures.

(4) Crystal structure of Aeropyrum pernix D-2-deoxyribose-5-phosphate aldolase
A gene encoding a D-2-deoxyribose-5-phosphate aldolase (DERA) homologuewas identified in the hyperthermophilic archaeon Aeropyrum pernix. The gene was　overexpressed in Escherichia coli, and the produced enzyme was purified and　characterized. The enzyme was an extremely thermostable DERA; the activity was　not lost after incubation at 100°C for 10 min. The enzyme had a molecular mass ofabout 93 kDa and consisted of four subunits with an identical molecular mass of 24 kDa.This shows the first presence of the tetrameric DERA. The three-dimensional　structure of the enzyme was determined by x-ray analysis (Fig. 3). Compared with thestructure of the E. coli DERA, the mainchain coordinate of the monomer of A. pernix　enzyme was quite similar to that of the E. coli enzyme. A large difference in　hydrophobic interactions and the number of ion pairs was not observed between themonomeric structures of the two enzymes. However, a significant difference in thequaternary structure was observed. The area of the subunit-subunit interface in thedimer of the A. pernix enzyme was much larger than the corresponding one of the E.coli enzyme. In addition, the A. pernix enzyme was 10 amino acids longer than the E.coli enzyme in the N-terminal region and exhibited an additional N-terminal helix.The N-terminal helix produced a unique dimer-dimer interface. This promotes theformation of a functional tetramer of the A. pernix enzyme and strengthens thehydrophobic inter-subunit interactions. These structural features are considered to beresponsible for the extremely high stability of the A. pernix enzyme
Protein Structural Gallery

Selected Papers
1) ”Structural basis of actin recognition and arginine ADP-rebosylation by Clostridium perfringens iota-toxin.”
Proc Natl Acad Sci U S A. 2008 May 19. [Epub ahead of print]
Tsuge, H., Nagahama, M., Oda, M., Iwamoto, S., Utsunomiya, H., Victor, E.M., Katunuma, N., Nishizawa M. and Sakurai J.
2) “Crystal structure of a novel FAD-,FMN-, and ATP-containing L-Proline Dehydrogenase Complex from Pyrococcus horikoshii.”
J. Biol. Chem. 2005 280(35):31045-31049
Tsuge, H., Kawakami, R., Sakuraba, H., Ago, H., Miyano, M., Aki, K., Katunuma, N. and Ohshima, T.
3) “Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin.”
J. Mol. Biol. 2003 325(3):471-483
Tsuge, H., Nagahama, M., Nishimura, H., Hisatsune, J., Sakaguchi, Y., Itogawa, Y., Katunuma, N. and Sakurai, J.
4) “Inhibition mechanism of cathepsin L-specific inhibitors based on the crystal structure of papain-CLIK148 complex.”
Biochem. Biophys. Res. Commun. 1999 266(2):411-416
Tsuge, H., Nishimura, T., Tada, Y., Asao, T., Turk, D., Turk, V. and Katunuma N.
5) "Structure of the Human Cytomegalovirus Protease Catalytic Domain Reveals a Novel Serine Protease Fold and Catalytic Triad."
Cell. 1996 86(5):835-843
Chen, P., Tsuge, H., Almassy, R.,Gribskov, C., Katoh, S., Vanderpool, C.,Margosiak, S., Pinko, C., Matthews, D., and Kan, CC.