Takeyuki Wakabayashi, Takuo Yasunaga, Takashi Ishikawa**, Yu Meng, Yoshiyuki Matsuura, Syuuhei Hashiba, Kimiko Saeki, Kazuo Sutoh* & Hitoshi Sasaki
Department of Physics, School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan and *Department of Life Science, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Tokyo 153, Japan , **RIKEN, Wako, Japan E-mail: wakabayashi@phys.s.u-tokyo.ac.jp
We examined the effect of mutagenesis introduced to the subdomain 4 of actin. Because the sequence of Gln228-Ser232 of Dictyostelium actin differs from that of Tetrahymena actin that does not bind tropomyosin, the Dicty/Tetra chimeric actin was produced. This mutant actin was functional. The chimeric actin (mutant 646: QTAAS-to-KAYKE replacement and E360H) showed decreased tropomyosin binding even in the presence of magnesium ions. This result indicates that the tropomyosin-binding sites of "on"-state actin are on the subdomain 4. The calcium-mediated regulation of actin-activated ATPase of S1 was analysed and it was found that the Hill coefficient for chimeric actin system was about 3 and that for control mutant was about 2. This result cannot be explained by steric blocking model and needs the aspect of cooperative/allosteric mechanism of thin filament regulation. We reconstructed three-dimensional structure of actin filaments with or without tropomyosin-troponin. The atomic model of actin was fitted well to our cryo-EM density maps. We calculated averaged density and standard deviation among about 20 three-dimensional images of filaments. We interpreted the standard deviation as the index of fluctuation of regions in actin monomers. MgADP-bound actin has rigid nucleotide-binding region, while the MgAMPPNP actin has the rigid region near the filament axis. It is interesting that subdomain-3 became more rigid after the release of gamma-phosphate, because it is assumed that subdomain-3 interacts with myosin. Moreover, MgADP-bound actin has the flexible hydrophobic plug region(262-272), which may result in the flexibility of actin filaments, which is observed macroscopically. With tropomyosin and troponin(I+C), we could observe the transconformation of thin filaments structure induced by calcium ions. Using holographic image reconstruction technique, the image of single myosin heads interacting with thin filaments were visualized.
REFERENCE Saeki, Sutoh & Wakabayashi(1996): Tropomyosin-Binding Site(s) on the Dictyostelium Actin Surface As Identified by Site-Directed Mutagenesis, Biochemistry, 35, 14465-14472
This work was supported by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Science and Culture of Japan, a Grant for "Biodesign Research Program" from the Institute of Physical Chemical Research(RIKEN) and a HFSP grant.