Protein Structural Biology Faculty of life Science* Kyoto Sangyo University**

Protein Structural Biology

Faculty of life Science*

Kyoto Sangyo University**

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 interaction. Latest research area was described below. We are collaborating with various biochemistry laboratories. We wish to provide important discovery based on crystal structures of biological macromolecules.

Please contact us (see member) if you are interested in our lab.

ADP-ribosylationg toxin and host human protein complex Clostridium perfringens type E produce ADP-ribosylating toxin (Iota toxin) 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 (JMB, Tsuge et al. 2003). 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. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog betaTAD at 2.8 A resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60-Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. (PNAS, Tsuge et al. (2008)) We would like to expand the understanding of these toxin and host factors. (See also “This week in PNAS”)

Influenza A virus RNA polymerase PB2 structure RNA-dependent RNA polymerase, comprising the PA, PB1, and PB2 subunits, is essential for viral transcription and replication. The binding of RNA primers/promoters to the polymerases is an initiation step in viral transcription. Lysine at position 627 in the polymerase in subunit PB2 are considered critical for the adaptation of avian influenza A viruses to mammals. We revealed the 2.7 A tertiary structure of the C-terminal RNA-binding domain of PB2 by x-ray crystallography. This domain incorporates lysine 627 of PB2 bearing the high pathogenicity and host range restriction of influenza A virus. We found from our current analyses that this lysine is located in a unique "phi"-shaped structure consisting of a helix and an encircled loop within the PB2 domain. Furthermore, by electrostatic analysis, we identified a highly basic groove along with this phi loop and found that lysine 627 is located in the phi loop. A PB2 domain mutant in which glutamic acid is substituted at position 627 shows significantly lower RNA binding activity. We would like to know the whole complex structure of RNA polymerase, PA, PB1, and PB2. This complex structure is expected for the structure based drug design of new influenza inhibitor. (JBC Kuzuhara et al. (2009))

Structure Based Drug Design of Cathepsin inhibitors 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 (See this paper). We determined the crystal structure of papain-CLIK148 complex and revealed the binding mode. The similarity and difference can be seen in the binding between CLIK148 and ZPACK.

Selected Papers

1) “Structural basis for the Helicobacter pylori-carcinogenic TNF-alpha-inducing protein”
Tsuge H, Tsurumura T, Utsunomiya H, Kise D, Kuzuhara T, Watanabe T, Fujiki H, Suganuma M.
Biochem Biophys Res Commun. 2009 Oct 16;388(2):193-8.

Helicobacter pylori-carcinogenic TNF-alpha-inducing protein forms the unique dimer structure, which has unique fold. The relationship between unique dimer structure and activity is still open question.

2) Structural basis of the influenza A virus RNA polymerase PB2 RNA-binding domain containing the pathogenicity-determinant residue lysine 627 residue.
Kuzuhara T, Kise D, Yoshida H, Horita T, Murazaki Y, Nishimura A, Echigo N, Utsunomiya H, Tsuge H.
J Biol Chem. 2009 Mar 13;284(11):6855-60. Epub 2009 Jan 14.

This paper provides not only the first crystal structure but also the RNA-binding activity of PB2 C-terminal domain which includes the human pathogenicity-determinant residue lysine 627.

3) ”Structural basis of actin recognition and arginine ADP-rebosylation by Clostridium perfringens iota-toxin.”
Proc Natl Acad Sci U S A. 2008 May 19.
Tsuge, H., Nagahama, M., Oda, M., Iwamoto, S., Utsunomiya, H., Victor, E.M., Katunuma, N., Nishizawa M. and Sakurai J.

This is the first complex structure of actin specific ADP-ribosylating toxin (ADPRT) and actin, It provides the novel information of the specificity of ADPRT and mechanism of the mono ADP-ribosylation.

4) “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.

This is an unique archaeal FAD-,FMN-, and ATP-containing enzyme and it consists of two subunit and three cofactors.

5) “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.

CLIK148-papain complex gave the important information of structure based drug design for cathepsins.

6) "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.

This paper describes the first crystal structure of Human Cytomegalovirus Protease,which is unique serine protease and provides the information for the structure based drug design.