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Our main focus is to reveal macromolecular structure of biologically important protein molecules using X-ray crystallography. Especially our interest is protein-protein complex and the interactions. Latest research area was described below. We are collaborating with various biochemistry laboratories. We wish to provide important discovery based on structural biology.

ADP-ribosylating toxin and substrate protein complex

Some bacterial protein toxins exert deleterious effects on target cells by entering cells and modifying an intracellular target. These toxins recognize a cellular receptor, translocate across the target cell membrane, and then exert their toxic effect against their targets.

Iota toxin, produced by Clostridium perfringens type E, is a member of the clostridial binary toxin group, which includes C. botulinum C2 toxin, C. difficile toxin (CDT), and C. spiroforme toxin (CST). These toxins are structurally related to the vegetative insecticidal proteins (VIP) produced by Bacillus cereus and B. thuringiensis (Barth et al. 2004). Recently, novel enterotoxins from non-CPE (C. perfringens enterotoxin) producing C. perfringens were found in humans. These toxins were named CPILE (C. perfringens iota-like enterotoxin) or BEC (binary enterotoxin of C. perfringens) (Yonogi et al. 2014; Monma et al. 2015; Irikura et al. 2015; Lyras and Rood 2014). All of these binary toxins consist of two unlinked proteins, A and B. The A component has ADP-ribosyltransferase (ART) activity toward actin, and the B component is involved in binding with a membrane receptor and translocation of the toxin into the cell. These ARTs ADP-ribosylate globular actin at Arg177, leading to the destruction of the filamentous actin and intoxicated cell.

In contrast, the Rho-specific C3-like ARTs produced by C. botulinum, C. limosum, B. cereus, and Staphylococcus aureus lack B components and are single domain enzymes (Sekine et al. 1989; Aktories et al. 1989). C. botulinum C3 (C3bot) ADP-ribosylates RhoA, RhoB, and RhoC at Asn41 and also modifies Rac1 very weakly. S. aureus C3 (C3aur) modifies RhoE and Rnd3 (Wilde et al. 2001). B. cereus C3 (C3cer) ADP-ribosylates RhoA, RhoB, and RhoC but does not modify Rac1 or Cdc42 at all (Wilde et al. 2003). Though Asn41 is conserved in Rac1 and Cdc42, they are not good substrates of C3-like toxins. Rho GTPases are master regulators of the actin cytoskeleton (Etienne-Manneville and Hall 2002), and their C3-catalyzed ADP-ribosylation causes their biological inactivation and thus the inhibition of downstream signaling and its consequences.

Comparative studies of these ARTs are very interesting. One reason is that their tertiary structures are very similar, but their targets are totally different. Recently, we revealed the crystal structure of an actin-specific ART (Ia) in complex with actin (Tsuge et al. 2008; Tsurumura et al. 2013). Furthermore, we also revealed the structure of the C3-RhoA complex (Toda et al. 2015). Comparison of the structures of these two complexes provided novel insight concerning substrate protein recognition and the mechanism of ADP-ribosylation. (See these two complex structures in top page)

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Aktories, K., Braun, U., Rosener, S., Just, I., and Hall, A.
The rho gene product expressed in E. Coli is a substrate of botulinum ADP-ribosyltransferase C3.
Biochem. Biophys. Res. Commun. 158, 209-213 (1989)

Barth, H., Aktories, K., Popoff, M. R., and Stiles, B. G.
Binary Bacterial Toxins: Biochemistry, Biology, and Applications of Common Clostridium and Bacillus Proteins.
Microbiol. Mol. Biol. Rev. 68, 373-402 (2004)

Etienne-Manneville, S., and Hall, A.
Rho GTPases in cell biology.
Nature. 420, 629-635 (2002)

Irikura, D., Monma, C., Suzuki, Y., Nakama, A., Kai, A., Fukui-Miyazaki, A., Horiguchi, Y., Yoshinari, T., Sugita-Konishi, Y., and Kamata, Y.
Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.
PLoS ONE. 10, e0138183 (2015)

Lyras, D., and Rood, J. I.
Preface: ClostPath 2013 meeting on The Molecular Biology and Pathogenesis of the Clostridia special issue.
Anaerobe. 30, 183 (2014)

Monma, C., Hatakeyama, K., Obata, H., Yokoyama, K., Konishi, N., Itoh, T., and Kai, A.
Four foodborne disease outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens.
J. Clin. Microbiol. 53, 859-867 (2015)

Sekine, A., Fujiwara, M., and Narumiya, S.
Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase.
J. Biol. Chem. 264, 8602-8605 (1989)

Toda, A., Tsurumura, T., Yoshida, T., Tsumori, Y., and Tsuge, H.
Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure.
J. Biol. Chem. 290, 19423-19432 (2015)

Tsuge, H., Nagahama, M., Oda, M., Iwamoto, S., Utsunomiya, H., Marquez, V. E., Katunuma, N., Nishizawa, M., and Sakurai, J.
Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens iota-toxin.
Proc. Natl. Acad. Sci. U.S.A. 105, 7399-7404 (2008)

Tsurumura, T., Tsumori, Y., Qiu, H., Oda, M., Sakurai, J., Nagahama, M., and Tsuge, H.
Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin and actin complex.
Proc. Natl. Acad. Sci. U.S.A. 110, 4267-4272 (2013)

Wilde, C., Chhatwal, G. S., Schmalzing, G., Aktories, K., and Just, I.
A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3.
J. Biol. Chem. 276, 9537-9542 (2001)

Wilde, C., Vogelsgesang, M., and Aktories, K.
Rho-specific Bacillus cereus ADP-ribosyltransferase C3cer cloning and characterization.
Biochemistry. 42, 9694-9702 (2003)

Yonogi, S., Matsuda, S., Kawai, T., Yoda, T., Harada, T., Kumeda, Y., Gotoh, K., Hiyoshi, H., Nakamura, S., Kodama, T., and Iida, T.
BEC, a novel enterotoxin of Clostridium perfringens found in human clinical isolates from acute gastroenteritis outbreaks.
Infect. Immun. 82, 2390-2399 (2014)