2023年05月, 海外日本人研究者ネットワーク, 2023年 UJA論文賞 (特別賞)
稲葉理美, 36800003

2022年12月, 日本分子生物学会, MBSJ2022 Science Pitch Award
Satomi Inaba-Inoue, 36800003

2022年04月, The British Crystallographic Association, BSG Group Poster Prize
Satomi Inaba-Inoue, 国際学会・会議・シンポジウム等の賞, グレートブリテン・北アイルランド連合王国(英国), 36800003

2022年04月, The British Crystallographic Association, BSG David Blow Prize (1st Prize)
Satomi Inaba-Inoue, 国際学会・会議・シンポジウム等の賞, グレートブリテン・北アイルランド連合王国(英国), 36800003

2021年05月, ASG-Keio, 第2回SXRハッカソン大賞 (1st Prize)　　　　　　　　　　　　　　　
小嶋良輔;赤木紀之;井上(稲葉)理美;大西里奈;合屋智尋;Davin HE Stetiamarga;甘利直花

2017年11月, 8th International and 10th Japan-China Joint Symposium on Calorimetry and Thermal Analysis, Best Poster Award　　　　　　　　　　　　　　　
Satomi Inaba

2017年07月, 日本農芸化学会 関西支部, 日本農芸化学会関西支部優秀発表賞　　　　　　　　　　　　　　　
稲葉理美

2016年03月, 京都府公立大学法人, 理事長表彰　　　　　　　　　　　　　　　
稲葉理美

2015年10月, 日本熱測定学会, 第51回 熱測定討論会 鳩山賞 (ポスター賞)　　　　　　　　　　　　　　　
稲葉理美

2012年12月, GEヘルスケアジャパン, Biasymposium 2012 優秀ポスター賞　　　　　　　　　　　　　　　
稲葉理美

Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications
Damon Griffiths, Malcolm Anderson, Keith Richardson, Satomi Inaba-Inoue, William J. Allen, Ian Collinson, Konstantinos Beis, Michael Morris, Kevin Giles, Argyris Politis
Analytical Chemistry, American Chemical Society (ACS), 2024年04月01日
英語, 研究論文（学術雑誌）, Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localisation of dynamic changes. Consequently, HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase data quality by providing an orthogonal mode of peptide ion separation in the gas-phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage and/or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multi-pass IM separations. Using 1-pass and multi-pass cIM routines, we use the recently commercialised SELECT SERIES™ Cyclic™ IM spectrometer for HDX-MS analyses of 4 detergent solubilised IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multi-pass cIM data. Interestingly, use of 1-pass and multi-pass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM which has the potential to enable the analysis of more complex systems with greater accuracy and speed.

Engineering Supramolecular Hybrid Architectures with Directional Organofluorine Bonds
Kotei, PA, Paley, DW, Oklejas, V, Mittan-Moreau, DW, Schriber, EA, Aleksich, M, Willson, MC, Inoue, I, Owada, S, Tono, K, Sugahara, M, Inaba-Inoue, S, Aquila, A, Poitevin, F, Blaschke, JP, Lisova, S, Hunter, MS, Sierra, RG, Gascón, JA, Sauter, NK, Brewster, AS, Hohman, JN
Small Science, 4, 1, Wiley, 2024年01月
英語, 研究論文（学術雑誌）, Understanding how chemical modifications alter the atomic‐scale organization of materials is of fundamental importance in materials engineering and the target of considerable efforts in computational prediction. Incorporating covalent and noncovalent interactions in designing crystals while “piggybacking” on the driving force of molecular self‐assembly has augmented efforts to understand the emergence of complex structures using directed synthesis. In this work, microcrystalline powders of the silver 2‐, 3‐, and 4‐fluorobenzenethiolates are prepared and their structures are resolved by small‐molecule serial femtosecond X‐ray crystallography. These three compounds enable the emergence and role of supramolecular synthons in the crystal structures of 3D metal‐organic chalcogenolates to be examined. The unique divergence in their optoelectronic, morphological, and structural behaviors is assessed. The extent of CHF interactions and their influence on the structure and the observed trends in the thermal stability of the crystals are quantified through theoretical calculations and thermogravimetric analysis.

Cyclic ion mobility for hydrogen/deuterium exchange-mass spectrometry applications
Damon Griffiths, Malcolm Anderson, Keith Richardson, Satomi Inaba-Inoue, William J. Allen, Ian Collinson, Konstantinos Beis, Michael Morris, Kevin Giles, Argyris Politis
2023年12月20日
Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localisation of dynamic changes. Consequently, HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase data quality by providing an orthogonal mode of peptide ion separation in the gas-phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage and/or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multi-pass IM separations. Using 1-pass and multi-pass cIM routines, we use the recently commercialised SELECT SERIES™ Cyclic™ IM spectrometer for HDX-MS analyses of 4 detergent solubilised IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multi-pass cIM data. Interestingly, use of 1-pass and multi-pass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM which has the potential to enable the analysis of more complex systems with greater accuracy and speed.

CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs
Piotr Stępień, Sylwia Świątek, Manuel Yamil Yusef Robles, Joanna Markiewicz-Mizera, Dhanasekaran Balakrishnan, Satomi Inaba-Inoue, Alex H. De Vries, Konstantinos Beis, Siewert J. Marrink, Jonathan G. Heddle
ACS Applied Materials & Interfaces, American Chemical Society (ACS), 2023年11月28日, ［査読有り］
研究論文（学術雑誌）

Functional Structure Analysis of DNA-binding Protein by SAXS
Satomi Inaba, Satomi Inaba-Inoue
SPring-8/SACLA Research Report, SPring-8/SACLA Research Report, 2023年10月31日
日本語, 研究論文（学術雑誌）

Cryogenic electron microscope facility at KEK-SBRC
Akihito Ikeda, Masato Kawasaki, Takayuki Kubota, Misato Yamamoto, Yusuke Yamada, Satomi Inaba-Inoue, Akira Takasu, Shinji Aramaki, Chiho Masuda, Naruhiko Adachi, Toshio Moriya, Toshiya Senda
Acta Crystallographica Section A Foundations and Advances, 2023年07月07日
研究論文（学術雑誌）

Dual-Uptake Mode of the Antibiotic Phazolicin Prevents Resistance Acquisition by Gram-Negative Bacteria
Dmitrii Y. Travin, Romain Jouan, Armelle Vigouroux, Satomi Inaba-Inoue, Joy Lachat, Fazal Haq, Tatiana Timchenko, Dmitry Sutormin, Svetlana Dubiley, Konstantinos Beis, Solange Moréra, Konstantin Severinov, Peter Mergaert
mBio, American Society for Microbiology, 2023年02月21日
研究論文（学術雑誌）, Many bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides act either by membrane disruption or by inhibiting essential intracellular processes.

The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria
Travin, DY, Vigouroux, A, Inaba-Inoue, S, Qu, F, Jouan, R, Lachat, J, Sutormin, D, Dubiley, S, Beis, K, Moréra, S, Severinov, K, Mergaert, P
Cold Spring Harbor Laboratory, 2022年04月28日
研究論文（学術雑誌）, ABSTRACTPhazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer Rhizobium sp. Pop5. Using genetic and biochemical techniques, we here identified BacA and YejABEF as two importers of PHZ in a sensitive model strain Sinorhizobium meliloti Sm1021. BacA and YejABEF are members of SLiPT and ABC transporter families of non-specific peptide importers, respectively. The uptake of PHZ by two distinct families of transporters dramatically decreases the naturally occurring rate of resistance. Moreover, since both BacA and YejABEF are essential for the development of functional symbiosis of rhizobia with leguminous plants, the acquisition of PHZ resistance via the inactivation of transporters is further disfavoured since single bacA or yejABEF mutants are unable to propagate in root nodules. Crystal structures of the periplasmic subunit YejA from S. meliloti and Escherichia coli revealed fortuitous bound peptides, suggesting a non-specific peptide-binding mechanism that facilitates the uptake of PHZ and other antimicrobial peptides.SIGNIFICANCEMany bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides kill bacteria by either membrane disruption or inhibiting essential intracellular processes. The Achilles heel of the latter type of antimicrobials is their dependence on transporters to enter the susceptible bacteria since mutations in such transporters result in resistance. We describe here how the ribosome-targeting peptide phazolicin, produced by Rhizobium sp. Pop5, uses two different transporters, BacA and YejABEF, to get into the cells of the symbiotic bacterium Sinorhizobium meliloti. This dramatically reduces the probability of resistance acquisition. Both transporters need to be inactivated for phazolicin resistance acquisition. Since these transporters are also crucial in S. meliloti for its symbiotic association with host plants, their inactivation in biological settings is highly unlikely. This makes PHZ an attractive lead for the development of a biocontrol agent with potential for use in agriculture.

Letters from Abroad
Satomi Inaba-Inoue
Seibutsu Butsuri, 62, 2, 148, 149, Biophysical Society of Japan, 2022年
英語, 研究論文（学術雑誌）

Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides
Dmitry Ghilarov, Satomi Inaba-Inoue, Piotr Stepien, Feng Qu, Elizabeth Michalczyk, Zuzanna Pakosz, Norimichi Nomura, Satoshi Ogasawara, Graham Charles Walker, Sylvie Rebuffat, So Iwata, Jonathan Gardiner Heddle, Konstantinos Beis
Science Advances, 7, 37, American Association for the Advancement of Science (AAAS), 2021年09月10日, ［査読有り］, ［筆頭著者］
研究論文（学術雑誌）, 36800003

CRYSTAL AND SOLUTION STRUCTURES OF SH2 DOMAIN OF SIGNALING MOLECULE IN COMPLEX WITH THE CO-STIMULATORY RECEPTOR CD28
Inaba-Inoue, S, Inoue, K, Hikima, T, Sekiguchi, H, Rambo, R
Acta Crystallographica Section A: Foundations and Advances, 75, a2, E69, E69, INT UNION CRYSTALLOGRAPHY, 2019年08月18日, ［筆頭著者］
研究論文（学術雑誌）

Site-specific observation of the conformational change of a protein with ¹⁵N-labeled Tyr residues using NMR
Inaba, S, Shiota, A, Yoshida, T, Oda, M
Analytical Biochemistry, 574, 34, 38, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2019年06月01日, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Binding thermodynamics of metal ions to HIV-1 ribonuclease H domain
Oda, M, Xi, Z, Inaba, S, Slack, RL, Ishima, R
Journal of Thermal Analysis and Calorimetry: an international forum for thermal studies, 135, 5, 2647, 2653, SPRINGER, 2019年03月
英語, 研究論文（学術雑誌）

Folding thermodynamics of PET-hydrolyzing enzyme Cut190 depending on Ca²⁺ concentration
Inaba, S, Kamiya, N, Bekker, G-J, Kawai, F, Oda, M
Journal of Thermal Analysis and Calorimetry: an international forum for thermal studies, 135, 5, 2655, 2663, SPRINGER, 2019年03月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Structural and functional properties of Grb2 SH2 dimer in CD28 binding
Yuhi Hosoe, Nobutaka Numoto, Satomi Inaba, Shuhei Ogawa, Hisayuki Morii, Ryo Abe, Nobutoshi Ito, Masayuki Oda
Biophysics and Physicobiology, 16, 80, 88, Biophysical Society of Japan, 2019年, ［国内誌］
英語, 研究論文（学術雑誌）, Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein that plays a critical role in cellular signal transduction. It contains a central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. Binding of Grb2 SH2 to the cytoplasmic region of CD28, phosphorylated Tyr (pY) containing the peptide motif pY-X-N-X, is required for costimulatory signaling in T cells. In this study, we purified the dimer and monomer forms of Grb2 SH2, respectively, and analyzed their structural and functional properties. Size exclusion chromatography analysis showed that both dimer and monomer exist as stable states. Thermal stability analysis using circular dichroism showed that the dimer mostly dissociates into the monomer around 50°C. CD28 binding experiments showed that the affinity of the dimer to the phosphopeptide was about three fold higher than that of the monomer, possibly due to the avidity effect. The present crystal structure analysis of Grb2 SH2 showed two forms; one is monomer at 1.15 Å resolution, which is currently the highest resolution analysis, and another is dimer at 2.00 Å resolution. In the dimer structure, the C-terminal region, comprising residues 123-152, was extended towards the adjacent molecule, in which Trp121 was the hinge residue. The stable dimer purified using size exclusion chromatography would be due to the C-terminal helix "swapping". In cases where a mutation caused Trp121 to be replaced by Ser in Grb2 SH2, this protein still formed dimers, but lost the ability to bind CD28.

Enzymatic hydrolysis of PET: functional roles of three Ca2+ ions bound to a cutinase-like enzyme, Cut190*, and its engineering for improved activity
Masayuki Oda, Yuri Yamagami, Satomi Inaba, Tatsuo Oida, Masaki Yamamoto, Sakihito Kitajima, Fusako Kawai
Applied Microbiology and Biotechnology, 102, 23, 10067, 10077, Springer Science and Business Media {LLC}, 2018年12月
英語, 研究論文（学術雑誌）, Cut190 from Saccharomonospora viridis AHK190 (Cut190) is the only cutinase that exhibits inactive (Ca2+-free) and active (Ca2+-bound) states, although other homologous cutinases always maintain the active states (Ca2+-free and bound). The X-ray crystallography of the S176A mutant of Cut190* (Cut190_S226P/R228S) showed that three Ca2+ ions were bound at sites 1–3 of the mutant. We analyzed the roles of three Ca2+ ions by mutation and concluded that they play different roles in Cut190* for activation (sites 1 and 3) and structural and thermal stabilization (sites 2 and 3). Based on these analyses, we elucidated the mechanism for the conformational change from the Ca2+-free inactive state to the Ca2+-bound active state, proposing the novel Ca2+ effect on structural dynamics of protein. The introduction of a disulfide bond at Asp250 and Glu296 in site 2 remarkably increased the melting temperatures of the mutant enzymes by more than 20–30 °C (while Ca2+-bound) and 4–14 °C (while Ca2+-free), indicating that a disulfide bond mimics the Ca2+ effect. Replacement of surface asparagine and glutamine with aspartic acid, glutamic acid, or histidine increased the melting temperatures. Engineered mutant enzymes were evaluated by an increase in melting temperatures and kinetic values, based on the hydrolysis of poly(butylene succinate-co-adipate) and microfiber polyethylene terephthalate (PET). A combined mutation, Q138A/D250C-E296C/Q123H/N202H, resulted in the highest thermostability, leading to the maximum degradation of PET film (more than 30%; approximately threefold at 70 °C, compared with that of Cut190* at 63 °C).

Effects of active site residues of 3-hydroxysteroid dehydrogenase from pseudomonas sp b-0831 on its catalysis and cofactor binding
Shiota, A, Inaba, S, Oda, M
Bioscience, Biotechnology and Biochemistry, 82, 10, 1702, 1707, TAYLOR \& FRANCIS LTD, 2018年10月03日
英語, 研究論文（学術雑誌）, ABSTRACT
We overexpressed and purified 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD) and its mutants where the active site residues known as the SYK triad, Ser114, Tyr153, and Lys157, were mutated. Ps3αHSD catalyzes the reaction by using a nucleotide cofactor. The NADH binding affinity of K157A mutant was much lower than that of the wild-type, mainly due to loss of a hydrogen bond. The decreased affinity would result in decreased kcat. Compared to the wild-type, the mutants S114A and Y153F showed higher Km and lower kcat values in both oxidation and reduction reactions. Simultaneous mutation of S114A and Y153F resulted in a significant decrease in kcat relative to the single mutant. These results are supported by the notion that Tyr153 is a catalytic base and Ser114 would be a substitute. Loss of hydrogen bonding with NADH upon the Y153F mutation resulted in increased enthalpy change, partially compensated by increased entropy change.

Structural Dynamics of the PET-Degrading Cutinase-like Enzyme from Saccharomonospora viridis AHK190 in Substrate-Bound States Elucidates the Ca2+-Driven Catalytic Cycle
Nobutaka Numoto, Narutoshi Kamiya, Gert-Jan Bekker, Yuri Yamagami, Satomi Inaba, Kentaro Ishii, Susumu Uchiyama, Fusako Kawai, Nobutoshi Ito, Masayuki Oda
Biochemistry, 57, 36, 5289, 5300, American Chemical Society ({ACS}), 2018年09月11日
英語, 研究論文（学術雑誌）

DNA-binding induced conformational change of c-Myb R2R3 analyzed using diffracted X-ray tracking
Yuhi Hosoe, Satomi Inaba, Hiroshi Sekiguchi, Yuji C. Sasaki, Masayuki Oda
Biochemical and Biophysical Research Communications, 503, 1, 338, 343, Elsevier {BV}, 2018年09月, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Previous structural analyses have shown that R2R3, the minimum unit of the DNA-binding domain of the transcriptional factor c-Myb, is largely flexible in solution, and changes to a more rigid structure upon DNA binding. In this study, we evaluated the structural dynamics using the diffracted X-ray tracking method, in correlation with DNA-binding abilities under different salt conditions, and compared them with the previous results. The resultant curve of the mean square angular displacements (MSD) clearly showed that the flexibility of R2R3 was decreased upon DNA binding, and the DNA-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry. The results of the MSD curves also indicate that the translational length reduces by approximately half upon DNA binding.

Structural and thermodynamic characterization of endo-1,3-β-glucanase: Insights into the substrate recognition mechanism
Oda, M, Inaba, S, Kamiya, N, Bekker, G-J, Mikami, B
BBA: Proteins and Proteomics, 1866, 3, 415, 425, ELSEVIER, 2018年03月
英語, 研究論文（学術雑誌）

Effect of a salt-bridge between inter-repeats on the 3D structure of the c-Myb DNA-binding domain revealed by thermodynamic analysis
Inaba, S, Fukada, H, Oda, M
Journal of Thermal Analysis and Calorimetry: an international forum for thermal studies, 131, 1, 335, 341, SPRINGER, 2018年01月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2
Usui, D, Inaba, S, Kamatari, YO, Ishiguro, N, Oda, M
Biochemical and Biophysical Research Communications, 490, 4, 1205, 1209, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2017年09月02日
英語, 研究論文（学術雑誌）

First observation of metal ion-induced structural fluctuations of α-helical peptides by using diffracted X-ray tracking
Daiki Usui, Satomi Inaba, Hiroshi Sekiguchi, Yuji C. Sasaki, Toshiki Tanaka, Masayuki Oda
Biophysical Chemistry, 228, 81, 86, Elsevier {BV}, 2017年09月, ［国際誌］
英語, 研究論文（学術雑誌）, In order to analyze protein structural dynamics, we designed simple model peptides whose structures changed from random-coil to helix-bundle structures by forming stable hydrophobic core in the presence of metal ions. The strategy involved destabilizing a de novo designed three helix-bundle protein by substituting the residues present in its hydrophobic core with histidine and small amino acids. The conformational changes of peptides induced upon binding of Zn2+ to histidine were analyzed using circular dichroism spectroscopy, which revealed peptides, HA and HG, to be good candidates for further analyses. The diffracted X-ray tracking experiments showed that the structural fluctuations of both HA and HG were suppressed upon binding of Zn2+. We succeeded in observing the differences in fluctuations of HA and HG in solution between random-coil like and helix-bundle structures. The metal-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry.

Tryptophan introduction can change -glucan binding ability of the carbohydrate-binding module of endo-1,3--glucanase
Miki, A, Inaba, S, Maruno, T, Kobayashi, Y, Oda, M
Bioscience, Biotechnology and Biochemistry, 81, 5, 951, 957, TAYLOR \& FRANCIS LTD, 2017年05月04日
英語, 研究論文（学術雑誌）, Abstract
Endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1 has a carbohydrate-binding module (CBM-DK) at the C-terminal side of a catalytic domain. Out of the imperfect tandem α-, β-, and γ-repeats in CBM-DK, the α-repeat primarily contributes to β-glucan binding. This unique feature is derived from Trp273 in α-repeat, whose corresponding residues in β- and γ-repeats are Asp314 and Gly358, respectively. In this study, we generated Trp-switched mutants, W273A/D314W, D270A/W273A/D314W, W273A/G358W, and D270A/W273A/G358W, and analyzed their binding abilities toward laminarioligosaccharides and laminarin. While the binding affinities of D270A/W273A and W273A mutants were either lost or much lower than that of the wild-type, those of Trp-switched mutants recovered, indicating that a Trp introduction in β- or γ-repeat can substitute the α-repeat by primarily contributing to β-glucan binding. Thus, we have successfully engineered a CBM-DK that binds to laminarin by a mechanism different from that of the wild-type, but with similar affinity.

Pronounced effect of hapten binding on thermal stability of an anti-(4-hydroxy-3-nitrophenyl)acetyl antibody possessing a glycine residue at position 95 of the heavy chain
Sato, Y, Inaba, S, Fukada, H, Azuma, T, Oda, M
Molecular Immunology, 85, 130, 136, PERGAMON-ELSEVIER SCIENCE LTD, 2017年05月
英語, 研究論文（学術雑誌）

生理的条件下で揺らぎの大きなDNA結合タンパク質の機能構造解析
稲葉 理美
生物物理, 57, 5, 257, 258, 一般社団法人 日本生物物理学会, 2017年, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins
Satomi Inaba, Nobutaka Numoto, Shuhei Ogawa, Hisayuki Morii, Teikichi Ikura, Ryo Abe, Nobutoshi Ito, Masayuki Oda
Journal of Biological Chemistry, 292, 3, 1052, 1060, Elsevier {BV}, 2017年01月, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.

Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl
Yusui Sato, Yusuke Tanaka, Satomi Inaba, Hiroshi Sekiguchi, Takahiro Maruno, Yuji C. Sasaki, Harumi Fukada, Yuji Kobayashi, Takachika Azuma, Masayuki Oda
International Journal of Biological Macromolecules, 91, 151, 157, Elsevier {BV}, 2016年10月, ［国際誌］
英語, 研究論文（学術雑誌）, Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.

Structural and binding properties of laminarin revealed by analytical ultracentrifugation and calorimetric analyses
Masayuki Oda, Yoichi Tanabe, Masanori Noda, Satomi Inaba, Elena Krayukhina, Harumi Fukada, Susumu Uchiyama
Carbohydrate Research, 431, 33, 38, Elsevier {BV}, 2016年08月
英語, 研究論文（学術雑誌）, One of the β-1,3-glucans, laminarin, has been widely used as a substrate for enzymes including endo-1,3-β-glucanase. To obtain quantitative information about the molecular interaction between laminarin and endo-1,3-β-glucanase, the structural properties of laminarin should be determined. The results from pioneering work using analytical ultracentrifugation for carbohydrate analysis showed that laminarin from Laminaria digitata predominantly exists as a single-chain species with approximately 5% of triple-helical species. Differential scanning calorimetry experiments did not show a peak assignable to the transition from triple-helix to single-chain, supporting the notion that a large proportion of laminarin is the single-chain species. The interaction of laminarin with an inactive variant of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, E119A, was quantitatively analyzed using isothermal titration calorimetry. The binding was enthalpically driven and the binding affinity was approximately 106 M-1. The results from binding stoichiometric analysis indicated that on average, E119A binds to laminarin in a 2:1 ratio. This seems to be reasonable, because laminarin mainly exists as a monomer, the apparent molecular mass of laminarin is 3.6 kDa, and E119A would have substrate-binding subsites corresponding to 6 glucose units. The analytical ultracentrifugation experiments could detect different complex species of laminarin and endo-1,3-β-glucanase.

Thermodynamic effects of a linker region between two repeats of a protein, c-Myb R2R3, on its stability and structural dynamics
Inaba, S, Fukada, H, Oda, M
Journal of Thermal Analysis and Calorimetry: an international forum for thermal studies, 123, 3, 1763, 1767, SPRINGER, 2016年03月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Folding thermodynamics of c-Myb DNA-binding domain in correlation with its α-helical contents
Inaba, S, Fukada, H, Oda, M
International Journal of Biological Macromolecules, 82, 725, 732, ELSEVIER SCIENCE BV, 2016年01月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Functional conformer of c-Myb DNA-binding domain revealed by variable temperature studies
Satomi Inaba, Akihiro Maeno, Kazumasa Sakurai, Sunilkumar Puthenpurackal Narayanan, Takahisa Ikegami, Kazuyuki Akasaka, Masayuki Oda
FEBS Journal, 282, 23, 4497, 4514, Wiley, 2015年12月, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The conformational fluctuation in the minimum DNA-binding domain of c-Myb, repeats 2 and 3 (R2R3), was studied under closely physiological conditions. A global unfolding transition, involving both the main chain and the side chains, was found to take place at the approximate temperature range 30-70 °C, with a transition temperature of approximately 50 °C. In addition, the observation of simultaneous shift change and broadening of NMR signals in both (1)H one-dimensional and (15)N/(1)H two-dimensional NMR spectra indicated the occurrence of locally fluctuating state at physiological temperature. In the wild-type protein containing a cavity in R2, the local fluctuation of R2 is more prominent than that of R3, whereas it is suppressed in the cavity-filled mutant, V103L. This indicates that the cavity in R2 contributes significantly to the conformational instability and the transition into the locally fluctuating state. For the wild-type R2R3 protein, the more dynamic conformer is estimated to be present to some extent at 37 °C and is likely beneficial for its biological function: DNA-binding. This result is in agreement with the concept of an excited-state conformer that exists in equilibrium with the dominant ground-state conformer and acts as the functional conformer of the protein. From the findings of the present study, it appears that the tandem repeats of two small domains with no disulfide bonds and with a destabilizing cavity function as the evolutionary strategy of the wide-type c-Myb DNA-binding domain to produce an appropriate fraction of the locally fluctuating state at 37 °C, which is more amenable to DNA-binding. Database: Chemical shifts and peak lists have been deposited in the Biological Magnetic Resonance Bank under entries 11584 and 11585.

Structural and physical properties of collagen extracted from moon jellyfish under neutral pH conditions
Miki, A, Inaba, S, Baba, T, Kihira, K, Fukada, H, Oda, M
Bioscience, Biotechnology and Biochemistry, 79, 10, 1603, 1607, TAYLOR \& FRANCIS LTD, 2015年10月03日, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）, Abstract
We extracted collagen from moon jellyfish under neutral pH conditions and analyzed its amino acid composition, secondary structure, and thermal stability. The content of hydroxyproline was 4.3%, which is lower than that of other collagens. Secondary structure analysis using circular dichroism (CD) showed a typical collagen helix. The thermal stability of this collagen at pH 3.0 was lower than those from fish scale and pig skin, which also correlates closely with jellyfish collagen having lower hydroxyproline content. Because the solubility of jellyfish collagen used in this study at neutral pH was quite high, it was possible to analyze its structural and physical properties under physiological conditions. Thermodynamic analysis using CD and differential scanning calorimetry showed that the thermal stability at pH 7.5 was higher than at pH 3.0, possibly due to electrostatic interactions. During the process of unfolding, fibrillation would occur only at neutral pH.

Thermodynamic effects of multiple protein conformations on stability and DNA binding
Inaba, S, Fukada, H, Ikegami, T, Oda, M
Archives of Biochemistry and Biophysics, 537, 2, 225, 232, ELSEVIER SCIENCE INC, 2013年09月15日, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Crystal Structures of Hereditary Vitamin D-Resistant Rickets-Associated Vitamin D Receptor Mutants R270L and W282R Bound to 1,25-Dihydroxyvitamin D3and Synthetic Ligands
Makoto Nakabayashi, Yoshito Tsukahara, Yukiko Iwasaki-Miyamoto, Mika Mihori-Shimazaki, Sachiko Yamada, Satomi Inaba, Masayuki Oda, Masato Shimizu, Makoto Makishima, Hiroaki Tokiwa, Teikichi Ikura, Nobutoshi Ito
Journal of Medicinal Chemistry, 56, 17, 6745, 6760, American Chemical Society ({ACS}), 2013年09月12日
英語, 研究論文（学術雑誌）

Structural and thermodynamic analysis of co-stimulation receptor CD28 phosphopeptide interactions with Grb2, Gads, and PI3-kinese SH2 domains
Satomi Inaba, Nobutaka Numoto, Hisayuki Morii, Teikichi Ikura, Ryo Abe, Nobutoshi Ito, Masayuki Oda, PROTEIN SCIENCE, 24, 287, 287, 2015年10月
WILEY-BLACKWELL, 英語, 研究発表ペーパー・要旨（国際会議）

Generation of single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl and analysis of its structural dynamics
Yusui Sato, Yusuke Tanaka, Hiroshi Sekiguchi, Satomi Inaba, Takahiro Maruno, Yuji C. Sasaki, Yuji Kobayashi, Takachika Azuma, Masayuki Oda, PROTEIN SCIENCE, 24, 163, 164, 2015年10月
WILEY-BLACKWELL, 英語, 研究発表ペーパー・要旨（国際会議）

細胞構造科学Ⅰ　　　　　　　　　　　　　　　
北海道大学

生体高分子学実験Ⅰ　　　　　　　　　　　　　　　
北海道大学

高分子機能学基礎実験　　　　　　　　　　　　　　　
北海道大学

一般教育演習 (フレッシュマンセミナー) 北大バイオ研究への招待　　　　　　　　　　　　　　　
北海道大学

生命分子化学実験 IV　　　　　　　　　　　　　　　
京都府立大学

生物物理化学　　　　　　　　　　　　　　　
京都府立大学

情報処理基礎演習　　　　　　　　　　　　　　　
京都府立大学

複数の抗菌ペプチドを輸送する膜タンパク質の基質認識特性と輸送過程の解明
科学研究費助成事業
2024年04月 - 2027年03月
稲葉 理美
日本学術振興会, 基盤研究(C), 大学共同利用機関法人高エネルギー加速器研究機構, 研究代表者, 24K08708

ナノ集光X線自由電子レーザーを利用した構造解析法の開発
科学研究費助成事業
2022年04月 - 2025年03月
井上 伊知郎, 稲葉 理美
日本学術振興会, 基盤研究(B), 国立研究開発法人理化学研究所, 22H03877

創薬を指向した補助刺激受容体CD28ファミリーとシグナル伝達分子の構造基盤解明
科学研究費助成事業
2018年04月 - 2024年03月
稲葉 理美
2022年6月より、海外渡航に伴って一時中断していた本課題を再開した。2022年度はSAXSなどで得られた実験データを元に、構造情報との相関づけを試みた。具体的には、SAXSなどで得られた溶液情報は、結晶構造を支持する結果となっているが、一部の(分解能)領域においては一致しないことが分かっていた。その原因としては、構造解析で用いたリガンドと溶液物性解析で用いるリガンドの鎖長が異なっていることが考えられた。そのため、比較する分子モデルを既存の構造情報をもとに新たに構築して比較することにした。さらに、溶媒条件(pHやイオン強度)を変えることでもSAXSの特性が変わることもわかった。以上のことより、よりダイナミクスにフォーカスした解析が必要であることが明らかとなり、次年度に向けてどのような解析を進めるべきかなどの指針を立てることができた。
日本学術振興会, 若手研究, 18K14395

抗菌ペプチドを取り込む膜タンパク質SbmAの立体構造と輸送機構の解明
海外特別研究員
2019年10月 - 2022年06月
井上(稲葉) 理美
日本学術振興会, インペリアルカレッジロンドン, 研究代表者

補助刺激受容体を介する免疫系シグナル伝達分子の構造基盤の解明　　　　　　　　　　　　　　　
学術研究助成
2018年06月 - 2019年03月
稲葉理美
公益財団法人ひょうご科学技術協会

高付加価値機能性素材創製を目指したクラゲ由来コラーゲンの物性解析　　　　　　　　　　　　　　　
若手研究者育成支援費
2016年04月 - 2017年03月
稲葉理美
京都府公立大学法人

ｃ－Ｍｙｂ ＤＮＡ結合ドメインの機能に着目した生物学的揺らぎの多角的解析
科学研究費助成事業
2015年04月 - 2017年03月
稲葉 理美
2016年度は、前年度にNMRや熱測定で得られたc-Myb DNA結合ドメインに関する知見をさらに多角的に評価するべく、1分子解析や高圧下での研究を進めた。1分子解析は、SPring-8の高輝度X線を用いた時分割測定（DXT）を適用し、DNA結合に伴う運動性変化をミリ秒～マイクロ秒で追跡した。測定にあたり、変異体の作製やプローブのラベル部位などの検討も進め、1分子系のアンサンブル量の運動性変化と多分子系での揺らぎの情報がよく相関することを明らかにした。加えて、DNA結合状態での揺らぎの減少が認められ、これはITCやDSCで得られたエントロピー変化量の結果と相関することも明らかとなった。
高圧解析では、ダイアモンドアンビルセルを用いて、1 GPaを超える圧力まで測定することで、圧力軸での安定性の差異を明らかにすることに成功した。対象タンパク質の変性圧力中間点は約800 MPaであり、蛍光やNMR測定条件下においてはフォールド構造内の揺らぎを検出可能であることも示した。また、変異体を用いた実験により、タンパク質内部のキャビティと部分モル体積変化（ΔV）との間に良い相関を見出した。これらの結果は、これまでの温度変化やpH変化により得られた揺らぎの情報とも一致する。現在、双方ともに論文執筆中である。
さらに、リピート間の構造安定性に着目した研究も進め、c-MybがDNA結合に2つのリピートが必須である裏付けをフォールディングに関わる熱力学量により実証した。
日本学術振興会, 特別研究員奨励費, 京都府立大学, 研究代表者, 15J03576

免疫系シグナル伝達分子と受容体CD28ファミリー細胞内領域との分子間相互作用解析　　　　　　　　　　　　　　　
大学院生研究奨励事業
2014年06月 - 2015年03月
稲葉理美
植田安也子学術振興基金, 研究代表者

高圧NMR法および蛍光スペクトル法によるタンパク質の準安定構造と分子間相互作用の解析　　　　　　　　　　　　　　　
植田安也子基金大学院生研究奨励事業
2012年06月 - 2013年03月
稲葉理美
財団法人京都府立大学学術振興会, 研究代表者

転写因子c-Myb DNA結合ドメインの準安定構造の検出と機能構造との相関解明　　　　　　　　　　　　　　　
若手研究者育成支援費
2011年06月 - 2012年03月
稲葉理美
京都府公立大学法人, 研究代表者