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Structure group

Toshiya Senda, Ph.D.

High Energy Accelerator Research Organization Institute of Materials Structure Science
​ Structural Biology Research Center


Introduction

 

The Structural Biology Research Center (SBRC) at KEK operates synchrotron beamlines (for protein crystallography and bioSAXS) and a cryogenic electron microscope (cryo-EM) with the help of leading scientists. As an inter-university research institute, we support the users of these facilities. At the same time, we are pursuing in-house research projects, including investigations into GTP metabolism, to answer the ultimate question in biology—namely, What is life? Our body is composed of more than ten trillion cells, each of which has countless molecules. These molecules comprise various biological networks, such as metabolic, signaling, and transcriptional networks. They also interact with one another, creating a network of networks. This complex structure of cells seems inevitable considering the multi-potent nature of cells and must be a key to the robust and resilient nature of life. At SBRC, we would like to understand how these characteristics of life emerged from the viewpoint of molecules. For this purpose, tertiary structural information of biological macromolecules is indispensable information. Ultimately, reductionism will not be sufficient to unravel the mystery of life. At the same time, wholism cannot describe life fully. Both views need to be integrated to bridge micro and macro phenomena. Moreover, while structural biology is an essential research field for bridging micro and macro phenomena, structural biologists must collaborate with scientists in the fields of cell biology, OMICS, informatics, and molecular evolution to further widen the scope of biology. At SBRC we enthusiastically welcome all workers in these fields: Please join us!


Our Techniques
 

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SBRC is a leading research center of structural biology in Japan. We operate five beamlines for protein crystallography (PX) and two beamlines for biological small-angle X-ray scattering (bioSAXS) at the Photon Factory. Using the PX beamlines and the MR-native SAD method, it is possible to solve the phase problem with the anomalous scattering from sulfur atoms [1]. It is no longer necessary to prepare Se-Met-substituted proteins to solve the protein structures. Within the field of BioSAXS, not only SEC-SAXS but also titration SAXS is possible. The bioSAXS data can also be analyzed using our original software. In addition to the beamlines, a 200kV cryo-EM was installed in 2018 and has been opened for academic and industrial users. SBRC also has a state-of-the-art wet laboratory with an automated crystallization robot [2]. Using these facilities, we have determined the ternary structures of biological macromolecules, including GTP-metabolism relevant molecules and their complexes. In addition to these techniques, it is possible to use hybrid methods in SBRC [3]. Experts in each technique lend support to users of our facilities, offering input and collaboration as needed to help users complete their projects.


GTP Project
 

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To unravel the overall structure of the biological networks, it is not enough to know the relationships between genotypes and phenotypes, because the functional complexities of proteins are substantially more extensive than those of genes. Due to the complex characters of proteins arising from their tertiary structures, dynamics, cellular localizations, interaction partners, and post-translational modifications, it is hardly possible to comprehensively understand their functions in cells from genetic information alone, even in the era of AlphaFold2. In addition, deletion of one protein (or gene) from cells removes several biochemical functions simultaneously, introducing ambiguities into the analysis of results. Such ambiguities hamper precise elucidation of the biological network. To reduce them, researchers have utilized mutant proteins and small molecular weight inhibitors (molecular tools) to probe the biological functions of the target protein(s). However, it is still difficult to regulate their biochemical functions one by one. We have studied GTP metabolism and discovered the first cellular GTP sensor, PI5P4Kβ [4]. Intriguingly, PI5P4Kβ can catalyze kinase reactions using ATP and GTP. Since the binding sites of ATP and GTP are nearly the same, it is difficult to separate the ATP- and GTP-dependent catalytic activities by means of simple mutations. However, separating the two activities was necessary to prove our hypothesis that PI5P4Kβ senses cellular GTP levels and causes cellular responses. To solve this problem, we used high-resolution structural information and designed a PI5P4Kβ mutant protein that only lacks GTP-dependent activity [5]. In this way, we successfully proved our hypothesis. This result demonstrates that fine regulation of the protein functions is possible with the atomic resolution structures. Inhibitors are also critical to analyze protein functions in cells. Since inhibitors can quickly inactivate target proteins, their biological effects are different from those of mutations on cells. In particular, mutant cells frequently show adaptations. One of the critical challenges of the inhibitor design is isotype specificity. Since gene duplication that creates isotypes seems to be an origin of the complexity of life [6], the functional variety of isotypes is an important target for our research. We are currently trying to develop isotype-specific inhibitors for PI5P4Ks. Our group would like to contribute to the creation of molecular tools by revealing the functional tertiary structures of biological macromolecules at atomic resolution. The tertiary structures of the target protein at work will enable us to design a high-performance molecular tool(s) that inhibits a specific biochemical function of the target protein. This type of molecular tool would allow us to unveil the biological function of the target protein. However, it is still hard to reveal downstream signaling/metabolic pathways of the target protein. To complete the map of the biological network, we need to collaborate with other life science researchers such as those in the fields of OMICS, bioinformatics, and molecular evolution. Without these collaborations, structural studies cannot be adequately integrated into biological investigations. However, in the context of such collaboration, the structural study becomes a powerful tool. Our GTP research team is ideally equipped for providing collaboration in integrated biological research and has revealed new biological functions and signaling pathways of the GTP sensor (in preparation). Since GTP is one of the central metabolites in the regulation of cellular activities, we believe that GTP metabolism is relevant to high-level biological phenomena, including behavior and psychological activities. Our structural studies should provide a basis for understanding and regulating high-level biological activities. As a result, our structure analysis team will have exciting opportunities for collaboration. Our current structural targets include PI5P4Ks, IMPDH, and V-ATPase, which are essential players in GTP metabolism. Since we are also trying to understand the evolution of these proteins, structural analyses of target proteins from various species are underway. While the crystal structures of these proteins have already been clarified, we are trying to comprehensively understand the functional structures of these proteins. We can use not only X-ray crystallography but also cryo-EM and bioSAXS to achieve this goal.


Automated structure analysis

 In addition to the GTP-related structural analysis, SBRC has developed automated methods for structural biology. We have a highly automated crystallization screening system, which can screen 800 conditions in 30 minutes. The auto-data collection system at the PX beamlines allows us to collect more than 100 datasets in one day.
We are also developing a structure analysis pipeline for fully automated structure determination. The development of these systems will enable high-throughput structural analysis, leading to the creation of molecular tools.
 


For young researchers
 

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At SBRC, you can learn basic techniques to determine the ternary structure of biological macromolecules, including protein purification. We also have the capacity to perform advanced techniques for X-ray crystallography, bioSAXS, and cryo-EM. The cryo-EM techniques are especially important, since they enable not only atomic-resolution structure determination but also observation of cells at nano-level resolution is possible. These efforts are expected to bridge the gap between the atomic-resolution structure of proteins and cellular organelles. Cryo-EM and cryo-electron tomography (cryo-ET) are thus becoming indispensable techniques for cell biology. While we are currently performing only single particle analysis with cryo-EM, we would like to introduce the cryo-ET technique in the near future. In these and other ways, obtaining the skills for structural biology is critical to success in the life sciences in the 21st century. Students at SBRC can also gain sufficient training to become beamline scientists in synchrotron facilities themselves. Indeed, students with sufficient skills as a beamline scientist can be employed in any region of the world. Automation with robotics and software development with AI are growing fields. Combining some of these systems will open new possibilities in both structural biology and industrial research.

[References]

[1] Kumano, T., Hori, S., Watanabe, S., Terashita, Y., Yu, H, Y., Hashimoto, Y., Senda, T., Senda, M. *, Kobayashi, M. * (2021) FAD-dependent C-glycoside-metabolizing enzymes in cDNAs: screening, characterization, and crystal structure analysis. Proc. Natl. Acad. Sci USA, in press.

[2] Kato, R., Hiraki, M., Yamada, Y., Tanabe, M., Senda, T. A (2021) fully automated crystallization apparatus for small protein quantities. Acta Crystallogr. F77, 29-36.

[3] Hayashi, T., Senda, M., Suzuki, N., Nishikawa, H., Ben C., Tang, C., Nagase, L., Inoue, K., Senda, T. * And Hatakeyama, M. * (2017) Differential mechanism for SHP2 binding and activation are exploited by geographically distinct Helicobacter pylori CagA oncoproteins. Cell Rep 20, 2876-2890 .; Mori, T., Kumano, T., He, H., Watanabe, S ., Senda, M., Moriya, T., Adachi, N., Hori, S., Terashita, Y., Kawasaki, M., Hashimoto, Y., Awakawa, T., Senda, T. *, Abe, I. * & Kobayashi, M. * (2021) C-Glycoside modulation in the gut and in nature: Identification, characterization, structural analyzes and distribution of CC bond-cleaving enzymes. Nat. Commun. In press.

[4] Sumita, K., Lo, Y.-H., Takeuchi, K., Senda, M., Kofuji, S., Ikeda, Y., Terakawa, J., Sasaki, M., Yoshino, H. , Majd, N., Zheng, Y., Kahoud, ER, Yokota, T., Emerling, BM, Asara, JM, Ishida, T., Locasale, JW, Daikoku, T., Anastasiou, D., Senda, T . * and Sasaki, AT * (2016) The lipid kinase PI5P4Kβ is an intracellular GTP sensor for metabolism and tumorigenesis. Mol Cell 61, 187-198.

[5] Takeuchi, K., Senda, M., Lo, YH., Kofuji, S., Ikeda Y., Sasaki, AT * and Senda T. * (2016) Structural reverse genetics study of PI5P4β-nucleotide complexes reveals the presence of the GTP bioenergetics system in mammalian cells. FEBS J, 283, 3556-3562.  

[6] Kawakami, E., Adachi, N. Senda, T. and Horikoshi, M. * (2017) Leading role of TBP in the establishment of complexity in eukaryotic transcription initiation systems. Cell Rep. 21, 3941-3956.


Grant in progress
 

Corona-based CREST (Research area "Creation of a technological base that contributes to coexistence with infectious diseases such as the new coronavirus by fusion of different fields")
English name: [Fundamental technologies for COVID-19] Creation of fundamental technologies by interdisciplinary research to coexist with infectious diseases including COVID-19

 

Research subject title "Development of virus replication inhibition technology by controlling GTP metabolism" (Representative: Senda)
English subject name: A novel strategy to inhibit viral replication by controlling GTP metabolism (PI: T. Senda)
2021.2.1-2024.3.31


Finished grant
 

Koyanagi Foundation
Three-dimensional structure analysis of the membrane-bound structure of the GTP sensor PI5P4Kβ (Representative: Senda)
Structural analysis of the membrane-bound form of PI5P4Kβ
2020.4.1-2021 9.30

GTP GEEKS is engaged in GTP metabolism research using various approaches such as biochemical and structural methods. Each lab accepts master's and doctoral students (and postdocs).
If you are interested in GTP study, those who are considering go to the master's doctoral feel free to Contact Please contact us from.

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