Proceedings of the National Academy of Sciences Acta Medica Okayama 0027-8424 119 43 2022 Structures and mechanisms of actin ATP hydrolysis e2122641119 EN Yusuke Kanematsu Graduate School of Information Sciences, Hiroshima City University Akihiro Narita Structural Biology Research Center, Graduate School of Science, Nagoya University Toshiro Oda Faculty of Health and Welfare, Tokai Gakuin University Ryotaro Koike Graduate School of Informatics, Nagoya University Motonori Ota Graduate School of Informatics, Nagoya University Yu Takano Graduate School of Information Sciences, Hiroshima City University Kei Moritsugu Graduate School of Medical Life Science, Yokohama City University Ikuko Fujiwara Graduate School of Science, Osaka City University Kotaro Tanaka Structural Biology Research Center, Graduate School of Science, Nagoya University Hideyuki Komatsu Department of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology Takayuki Nagae Synchrotron Radiation Research Center, Nagoya University Nobuhisa Watanabe Synchrotron Radiation Research Center, Nagoya University Mitsusada Iwasa Graduate School of Informatics, Nagoya University Yuichiro Maéda Research Institute for Interdisciplinary Science, Okayama University Shuichi Takeda Research Institute for Interdisciplinary Science, Okayama University The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG–O3B bond cleavage; 3) four concomitant events: W1–PO3− formation, OH− and proton cleavage, nucleophilic attack by the OH− against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi–bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2296-634X 11 2023 Mutagenic analysis of actin reveals the mechanism of His161 flipping that triggers ATP hydrolysis 1105460 EN Mitsusada Iwasa Graduate School of Informatics, Nagoya University Shuichi Takeda Research Institute for Interdisciplinary Science (RIIS), Okayama University Akihiro Narita Structural Biology Research Center, Graduate School of Science, Nagoya University Yuichiro Maeda Research Institute for Interdisciplinary Science (RIIS), Okayama University Toshiro Oda Faculty of Health and Welfare, Tokai Gakuin University The dynamic assembly of actin is controlled by the hydrolysis of ATP, bound to the center of the molecule. Upon polymerization, actin undergoes a conformational change from the monomeric G-form to the fibrous F-form, which is associated with the flipping of the side chain of His161 toward ATP. His161 flipping from the gauche-minus to gauche-plus conformation leads to a rearrangement of the active site water molecules, including ATP attacking water (W1), into an orientation capable of hydrolysis. We previously showed that by using a human cardiac muscle a-actin expression system, mutations in the Pro-rich loop residues (A108G and P109A) and in a residue that was hydrogen-bonded to W1 (Q137A) affect the rate of polymerization and ATP hydrolysis. Here, we report the crystal structures of the three mutant actins bound to AMPPNP or ADP-P-i determined at a resolution of 1.35-1.55( )angstrom, which are stabilized in the F-form conformation with the aid of the fragmin F1 domain. In A108G, His161 remained non-flipped despite the global actin conformation adopting the F-form, demonstrating that the side chain of His161 is flipped to avoid a steric clash with the methyl group of A108. Because of the non-flipped His161, W1 was located away from ATP, similar to G-actin, which was accompanied by incomplete hydrolysis. In P109A, the absence of the bulky proline ring allowed His161 to be positioned near the Pro-rich loop, with a minor influence on ATPase activity. In Q137A, two water molecules replaced the side-chain oxygen and nitrogen of Gln137 almost exactly at their positions; consequently, the active site structure, including the W1 position, is essentially conserved. This seemingly contradictory observation to the reported low ATPase activity of the Q137A filament could be attributed to a high fluctuation of the active site water. Together, our results suggest that the elaborate structural design of the active site residues ensures the precise control of the ATPase activity of actin. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2399-3642 5 1 2022 Structural and biochemical evidence for the emergence of a calcium-regulated actin cytoskeleton prior to eukaryogenesis 890 EN Caner Akil Research Institute for Interdisciplinary Science (RIIS), Okayama University Linh T. Tran Research Institute for Interdisciplinary Science (RIIS), Okayama University Magali Orhant-Prioux CytomorphoLab, Biosciences & Biotechnology Institute of Grenoble, Laboratoire de Physiologie Cellulaire & Végétale, Université Grenoble-Alpes/CEA/CNRS/INRA Yohendran Baskaran Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Biopolis Yosuke Senju Research Institute for Interdisciplinary Science (RIIS), Okayama University Shuichi Takeda Research Institute for Interdisciplinary Science (RIIS), Okayama University Phatcharin Chotchuang School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC) Duangkamon Muengsaen School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC) Albert Schulte School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC) Edward Manser Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Biopolis Laurent Blanchoin CytomorphoLab, Biosciences & Biotechnology Institute of Grenoble, Laboratoire de Physiologie Cellulaire & Végétale, Université Grenoble-Alpes/CEA/CNRS/INRA Robert C. Robinson Research Institute for Interdisciplinary Science (RIIS), Okayama University Charting the emergence of eukaryotic traits is important for understanding the characteristics of organisms that contributed to eukaryogenesis. Asgard archaea and eukaryotes are the only organisms known to possess regulated actin cytoskeletons. Here, we determined that gelsolins (2DGels) from Lokiarchaeota (Loki) and Heimdallarchaeota (Heim) are capable of regulating eukaryotic actin dynamics in vitro and when expressed in eukaryotic cells. The actin filament severing and capping, and actin monomer sequestering, functionalities of 2DGels are strictly calcium controlled. We determined the X-ray structures of Heim and Loki 2DGels bound actin monomers. Each structure possesses common and distinct calcium-binding sites. Loki2DGel has an unusual WH2-like motif (LVDV) between its two gelsolin domains, in which the aspartic acid coordinates a calcium ion at the interface with actin. We conclude that the calcium-regulated actin cytoskeleton predates eukaryogenesis and emerged in the predecessors of the last common ancestor of Loki, Heim and Thorarchaeota. Calcium-regulated actin filament assembly predates eukaryogenesis and was present in the last common ancestor of Asgard archaea Loki, Heim, and Thorarchaeota. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0022-2836 433 9 2021 Structural Insights into the Regulation of Actin Capping Protein by Twinfilin C-terminal Tail 166891 EN Shuichi Takeda Research Institute for Interdisciplinary Science (RIIS), Okayama University Ryotaro Koike Graduate School of Informatics, Nagoya University Ikuko Fujiwara Graduate School of Science, Osaka City University Akihiro Narita Graduate School of Science, Nagoya University Makoto Miyata Graduate School of Science, Osaka City University Motonori Ota Graduate School of Informatics, Nagoya University Yuichiro Maéda Graduate School of Informatics, Nagoya University Twinfilin is a conserved actin regulator that interacts with actin capping protein (CP) via C-terminus residues (TWtail) that exhibits sequence similarity with the CP interaction (CPI) motif of CARMIL. Here we report the crystal structure of TWtail in complex with CP. Our structure showed that although TWtail and CARMIL CPI bind CP to an overlapping surface via their middle regions, they exhibit different CP-binding modes at both termini. Consequently, TWtail and CARMIL CPI restrict the CP in distinct conformations of open and closed forms, respectively. Interestingly, V-1, which targets CP away from the TWtail binding site, also favors the open-form CP. Consistently, TWtail forms a stable ternary complex with CP and V-1, a striking contrast to CARMIL CPI, which rapidly dissociates V-1 from CP. Our results demonstrate that TWtail is a unique CP-binding motif that regulates CP in a manner distinct from CARMIL CPI. No potential conflict of interest relevant to this article was reported.