Nature Portfolio Acta Medica Okayama 2399-3642 7 1 2024 The eukaryotic-like characteristics of small GTPase, roadblock and TRAPPC3 proteins from Asgard archaea 273 EN Linh T. Tran Research Institute for Interdisciplinary Science, Okayama University Caner Akil Research Institute for Interdisciplinary Science, Okayama University Yosuke Senju Research Institute for Interdisciplinary Science, Okayama University Robert C. Robinson Research Institute for Interdisciplinary Science, Okayama University Membrane-enclosed organelles are defining features of eukaryotes in distinguishing these organisms from prokaryotes. Specification of distinct membranes is critical to assemble and maintain discrete compartments. Small GTPases and their regulators are the signaling molecules that drive membrane-modifying machineries to the desired location. These signaling molecules include Rab and Rag GTPases, roadblock and longin domain proteins, and TRAPPC3-like proteins. Here, we take a structural approach to assess the relatedness of these eukaryotic-like proteins in Asgard archaea, the closest known prokaryotic relatives to eukaryotes. We find that the Asgard archaea GTPase core domains closely resemble eukaryotic Rabs and Rags. Asgard archaea roadblock, longin and TRAPPC3 domain-containing proteins form dimers similar to those found in the eukaryotic TRAPP and Ragulator complexes. We conclude that the emergence of these protein architectures predated eukaryogenesis, however further adaptations occurred in proto-eukaryotes to allow these proteins to regulate distinct internal membranes. No potential conflict of interest relevant to this article was reported.

American Association for the Advancement of Science (AAAS) Acta Medica Okayama 2375-2548 8 12 2022 Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution eabm2225 EN Caner Akıl Research Institute for Interdisciplinary Science, Okayama University Samson Ali Research Institute for Interdisciplinary Science, Okayama University Linh T. Tran Research Institute for Interdisciplinary Science, Okayama University Jérémie Gaillard University of Grenoble-Alpes, CEA, CNRS, INRA, Interdisciplinary Research Institute of Grenoble, Laboratoire de Physiologie Cellulaire & Végétale, CytoMorpho Lab Wenfei Li National Laboratory of Solid State Microstructure, Department of Physics, Collaborative Innovation Center of Advanced Microstructures, Nanjing University Kenichi Hayashida Cellular and Structural Physiology Institute (CeSPI), Nagoya University Mika Hirose Institute for Protein Research, Osaka University Takayuki Kato Institute for Protein Research, Osaka University Atsunori Oshima Cellular and Structural Physiology Institute (CeSPI), Nagoya University Kosuke Fujishima Tokyo Institute of Technology, Earth-Life Science Institute (ELSI) Laurent Blanchoin University of Grenoble-Alpes, CEA, CNRS, INRA, Interdisciplinary Research Institute of Grenoble, Laboratoire de Physiologie Cellulaire & Végétale, CytoMorpho Lab Akihiro Narita Division of Biological Science, Graduate School of Science, Nagoya University Robert C. Robinson Research Institute for Interdisciplinary Science, Okayama University Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound x-ray structures of an Asgard tubulin from hydrothermal living Odinarchaeota (OdinTubulin). The guanosine 5′-triphosphate (GTP)–bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the “+” end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolutionary stage intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins. 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 Inc Acta Medica Okayama 00219258 297 2021 A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria 101071 EN Yoshihito Kitaoku School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC) Tamo Fukamizo School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC) Sawitree Kumsaoad School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC) Prakayfun Ubonbal School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC) Robert C. Robinson Research Institute for Interdisciplinary Science, Okayama University Wipa Suginta School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC) VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)2 across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)2, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)3–4]. (GlcNAc)4 partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp363, Asp365, and Trp513) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)2, indicating their significant contributions to sugar binding. The structure of the W513A–(GlcNAc)2 complex in a ‘half-open’ conformation unveiled the intermediary step of the (GlcNAc)2 translocation from the soluble CBP in the periplasm to the inner membrane–transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)2, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients. No potential conflict of interest relevant to this article was reported.

American Association for the Advancement of Science Acta Medica Okayama 2375-2548 7 5 The structure of the actin filament uncapping complex mediated by twinfilin eabd5271 EN Dennis M. Mwangangi Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research) Edward Manser Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research) Robert C. Robinson Research Institute for Interdisciplinary Science (RIIS), Okayama University Uncapping of actin filaments is essential for driving polymerization and depolymerization dynamics from capping protein–associated filaments; however, the mechanisms of uncapping leading to rapid disassembly are unknown. Here, we elucidated the x-ray crystal structure of the actin/twinfilin/capping protein complex to address the mechanisms of twinfilin uncapping of actin filaments. The twinfilin/capping protein complex binds to two G-actin subunits in an orientation that resembles the actin filament barbed end. This suggests an unanticipated mechanism by which twinfilin disrupts the stable capping of actin filaments by inducing a G-actin conformation in the two terminal actin subunits. Furthermore, twinfilin disorders critical actin-capping protein interactions, which will assist in the dissociation of capping protein, and may promote filament uncapping through a second mechanism involving V-1 competition for an actin-binding surface on capping protein. The extensive interactions with capping protein indicate that the evolutionary conserved role of twinfilin is to uncap actin filaments. No potential conflict of interest relevant to this article was reported.

American Society for Biochemistry and Molecular Biology Acta Medica Okayama 0021-9258 295 14 2020 In vivo crystals reveal critical features of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and the PDZ2 domain of Na+/H+ exchange cofactor NHERF1 4464 4476 EN Eleanor R. Martin School of Biological Sciences, Faculty of Biology Medicine and Health, Michael Smith Building, The University of Manchester Alessandro Barbieri School of Biological Sciences, Faculty of Biology Medicine and Health, Michael Smith Building, The University of Manchester Robert C. Ford School of Biological Sciences, Faculty of Biology Medicine and Health, Michael Smith Building, The University of Manchester Robert C. Robinson Research Institute for Interdisciplinary Science, Okayama University Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)–PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions －1 and －3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice. No potential conflict of interest relevant to this article was reported.

National Academy of Sciences Acta Medica Okayama 0027-8424 2020 Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea 117 33 EN Caner Akıl Institute of Molecular and Cell Biology, Agency for Science, Technology and Research Linh T. Tran Research Institute for Interdisciplinary Science, Okayama University Magali Orhant-Prioux CytomorphoLab, Interdisciplinary Research Institute of Grenoble Yohendran Baskaran aInstitute of Molecular and Cell Biology, Agency for Science, Technology and Research Edward Manser aInstitute of Molecular and Cell Biology, Agency for Science, Technology and Research Laurent Blanchoin CytomorphoLab, Interdisciplinary Research Institute of Grenoble Robert C. Robinson cResearch Institute for Interdisciplinary Science, Okayama University Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1356-9597 25 1 2020 Tree of motility : A proposed history of motility systems in the tree of life 6 21 EN Makoto Miyata Department of Biology, Graduate School of Science, Osaka City University Robert C. Robinson Research Institute for Interdisciplinary Science, Okayama University Taro Q. P. Uyeda Department of Physics, Faculty of Science and Technology, Waseda University Yoshihiro Fukumori Faculty of Natural System, Institute of Science and Engineering, Kanazawa University Shun‐ichi Fukushima Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University Shin Haruta Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University Michio Homma Division of Biological Science, Graduate School of Science, Nagoya University Kazuo Inaba Shimoda Marine Research Center, University of Tsukuba Masahiro Ito Graduate School of Life Sciences, Toyo University Chikara Kaito Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo Kentaro Kato Laboratory of Sustainable Animal Environment, Graduate School of Agricultural Science, Tohoku University Tsuyoshi Kenri Laboratory of Mycoplasmas and Haemophilus, Department of Bacteriology II, National Institute of Infectious Diseases Yoshiaki Kinosita Department of Physics, Oxford University Seiji Kojima Division of Biological Science, Graduate School of Science, Nagoya University Tohru Minamino Graduate School of Frontier Biosciences, Osaka University Hiroyuki Mori Institute for Frontier Life and Medical Sciences, Kyoto University Shuichi Nakamura Department of Applied Physics, Graduate School of Engineering, Tohoku University Daisuke Nakane Department of Physics, Gakushuin University Koji Nakayama Department of Microbiology and Oral Infection, Graduate School of Biomedical Sciences, Nagasaki University Masayoshi Nishiyama Department of Physics, Faculty of Science and Engineering, Kindai University Satoshi Shibata Molecular Cryo‐Electron Microscopy Unit, Okinawa Institute of Science and Technology Graduate University Katsuya Shimabukuro Department of Chemical and Biological Engineering, National Institute of Technology, Ube College Masatada Tamakoshi Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences Azuma Taoka Faculty of Natural System, Institute of Science and Engineering, Kanazawa University Yosuke Tashiro Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University Isil Tulum Department of Botany, Faculty of Science, Istanbul University Hirofumi Wada Department of Physics, Graduate School of Science and Engineering, Ritsumeikan University Ken‐ichi Wakabayashi Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement‐producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility. No potential conflict of interest relevant to this article was reported.