3rd Joint International Symposium of
National Synchrotron Radiation Research Center, Taiwan
Institute for Protein Research, Osaka University, Japan

INSTITUTE for

PROTEIN RESEARCHOSAKA UNIVERSITY

Establishment of Structural Biology Network in Asia and Oceania
National Synchrotron Radiation Research Center
Hsinchu, Taiwan
May 21 - 22, 2024

Symposium Booklet

https://www.nsrrc.org.tw

http://www.protein.osaka-u.ac.jp

Joint International Symposium of NSRRC and IPR
Establishment of Structural Biology Nerwork in Asia
Rm. D260, National Synchrotron Radiation Research CenterMay 21 - 22, 2024
2024/5/21

10:00
10:15
0:15 Opening remarks & Brief of NSRRC
10:15
10:25
0:10 Opening remarks & Introduction of IPR
Session 1 Beamlines (Chair: Yu-Yuan Hsiao)
10:25
10:55
0:30 The current status of protein crystallography beamlines at the
NSRRC
10:55
11:15

11:15
11:45

0:20 Break (group photo @ 1st floor)
0:30 SPring-8 BL44XU, a beamline for large biologicalmacromolecular assemblies

11:45

12:15

0:30 Compound screening pipeline at SPring-8 macromolecular
crystallography beamlines for drug development

12:15
13:50
1:35 Lunch (NSRRC cafeteria) & TPS tour
Session 2 Fighting against diseases (Chair: Sachiko Toma-Fukai)
13:50
14:20
0:30 Anti-cancer drug discovery facilitated by X-ray protein
crystallography

Der-Hsin Wei
Genji Kurisu
Chun-Hsiang Huang

NSRRC, Taiwan
IPR, Japan
NYCU, Taiwan
NSRRC, Taiwan

Atsushi Nakagawa

IPR, Japan

Naoki Sakai

JASRI, Japan

Su-Ying Wu

NHRI, Taiwan

14:20

14:50

0:30 Structural basis for Rab1 glucosylation by Legionella SetA on Hyoun Sook Kim
the early Legionella -containing vacuole

NCC, Korea

14:50

15:20

0:30 Discovery and characterization of genes conferring resistance to Chin-Yuan Chang
antituberculosis antibiotic capreomycin

NYCU, Taiwan

15:20
15:40
0:20 BreakSession 3 molecular regulation, transportation, and proteolysis (Chair: Chin-Yuan Chang)
15:40
16:10
0:30 Cryo-EM structures of KtrAB reveal the synergism of ATP and Nien-Jen Hu
Na+ in activating the bacterial K+ uptake system

2024/5/22

16:10

16:40

0:30 Structural study of the nuclear transportation mechanism of
PPARγ by Transportin-1

16:40
17:10

17:10
17:40

0:30 Molecular basis of Cep57 in centrosome regulation
Hui-Chun Cheng
0:30 Structural basis for intramembrane proteolysis by bacterial site- Terukazu Nogi
2 proteases

17:40
18:00

18:00
20:00

0:20 Free time
2:00 Speaker banquet/dinner

Session 4 Structural dynamics with a hybrid approach (Chair: Terukazu Nogi)
9:50
10:20
0:30 Structure of cyanobacterial photosystem I complexed withferredoxin and cytochrome c6 at 1.97 Å resolution

Sachiko Toma-Fukai

NCHU, Taiwan
NAIST, Japan
NTHU, Taiwan
YCU, Japan

Genji Kurisu

IPR, Japan

10:20

10:50

0:30 Versatility of MarR domain in the regulation of phenolic
compound utilization

Danaya Pakotiprapha

MU, Thailand

10:50

11:20

0:30 Deciphering the calcium-dependent activation mechanism ofVibrio campbellii α-hemolysin

Shih-Ming Lin

NCKU, Taiwan

11:20
11:40

11:40
13:00

0:20 General Discussion and Conclusion
1:20 Lunch (NSRRC cafeteria)

Der-Hsin WEI
Deputy Director/Research Scientist
Directorate
National Synchrotron Radiation Research Center
E-mail: dhw@nsrrc.org.tw
BIOGRAPHY:
2022/08-present: Deputy Director at NSRRC, Taiwan
2014-present: Research Scientist at NSRRC, Taiwan
2012-present: Joint Appointment, Graduate Program for Science and Technology of SynchrotronLight Source, National Tsing Hua Univ., Taiwan
2012-present: Joint Appointment, Dept. of Physics, National Sun Yat-Sen Univ., Taiwan
2004-2013: Associate Research Scientist at NSRRC, Taiwan
2000-2003: Assistant Research Scientist at NSRRC, Taiwan
1999-2000: Visiting Scientist at ALS, US
1997-1999: Postdoctoral Fellow at NSRRC
1996-1997: Postdoctoral Fellow, IAMS, Academia Sinica, Taiwan

1Genji KURISU
Professor
Institute for Protein Research
Osaka University
E-mail: gkurisu@protein.osaka-u.ac.jp
BIOGRAPHY:
2024-present: Director, Institute for Protein Research, Osaka Univ., Japan2017-present: Head, Protein Data Bank Japan (PDBj), Osaka Univ., Japan
2009-present: Professor, Institute for Protein Research, Osaka Univ., Japan
2004-2009: Associate Professor, Department of Life Sciences, Univ. Tokyo, Japan
2002-2003: Visiting Scientist, Department of Biological Sciences, Purdue Univ., USA
1997-2004: Assistant Professor, Institute for Protein Research, Osaka Univ., Japan

Structure of cyanobacterial photosystem I complexed with ferredoxin andcyrtochrome c6 at 1.97 Å resolutionaGenji Kurisua,bLaboratory of Protein Crystallography, Institute for Protein Research, Osaka Universityb
Laboratory of Protein Databases, Institute for Protein Research, Osaka University

Cryo-EM structure of the trimeric Photosystem I (PSI) from thermophilic cyanobacterium,
Thermosynechococcus elongatus BP-1, complexed with its two electron transfer partner proteinsof Cytochrome c6 (Cyt c6) and Ferredoxin (Fd), has been solved to the highest record of 1.97 Å
resolution [1], thus revealing the specific molecular interaction of its donor and acceptor proteins.
Each protomer contains newly assigned seventeen lipids, three pigments, additional forty residues,and six water molecules at the interface with Fd. Fd-binding to PSI was thermodynamicallycharacterized also by Isothermal Titration Calorimetry measurement, which was complementary
to and consistent with cryo-EM analysis. The cryo-EM densities for Cyt c6 in our study are the firstexperimental result for visualization of Cyt c6 bound to PSI, even though the densities themselves
were not so intense, which might be induced by productive binding of Fd on the opposite side of
PSI. Based on all our new findings, a safe and productive electron transfer mechanism
accompanying two-way trans-membrane communications within the cyanobacterial PSI was
proposed.
Reference[1] Li, J., Hamaoka, N., Makino, F., Kawamoto, A., Lin, Y., Rögner, M., Nowaczyk, M.M., Lee,
Y.-H., Namba, K., Gerle, C., Kurisu, G, Commun Biol, 5 (1), 951 (2022)

1

Chun-Hsiang HUANG
Assistant Research Scientist
Biological Crystallography Facility
National Synchrotron Radiation Research Center (NSRRC)
E-mail: huang.conann@nsrrc.org.tw
BIOGRAPHY:
2015-present: Assistant Research Scientist, Biological Crystallography Facility, NSRRC.,
Hsinchu, Taiwan
2010-2014: Associate Research Fellow, Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Science, Tianjin, China
2009-2010: Postdoctoral Fellow, Genomics Research Center, Academia Sinica, Taiwan

The Current Status of Protein Crystallography Beamlines at the NSRRC
Chun-Hsiang Huang, Chung-Kuang Chou, Chien-Chang Tseng, Cheng-Hung Chiang,
Yi-Hui Chen, Yi-Chun Liu and Chun-Hsiung Chao*
National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan, ROC

The crystallographic analysis of proteins offers vital insights for life science researchers. To
date, protein crystallography remains a highly beneficial experimental method for determining
the structures of biological macromolecules. In addition to its significant academic value, its
demand in the industry is also increasing due to its highly automated characteristics. Recently,
several tough research projects, such as membrane and viral protein, have encountered challenges.
Specifically, crystals from these studies often fail to yield analyzable diffraction data whencollected at the TLS (Taiwan Light Source) protein beamlines, primarily due to their smaller sizeand large unit cells. To overcome these issues, two specialized TPS (Taiwan Photon Source)
beamlines have been constructed: the Protein Microcrystallography Beamline (TPS 05A),
designed for crystals with large unit cells, and the Micro-focus Protein Crystallography Beamline(TPS 07A), tailored for micro-crystals and inhomogeneous crystals. In the near future, there areplans to upgrade TPS 05A to enhance its capability for serial crystallography, thereby providing asolution for studying hard-to-handle crystals. In summary, three dedicated protein beamlines,
TLS 15A1, TPS 05A and TPS 07A, are currently available for the wide user base.

1

Atsushi NAKAGAWA
Professor
Institute for Protein Research
Osaka University
E-mail: atsushi@protein.osaka-u.ac.jp
BIOGRAPHY:
2003-present: Professor, Institute for Protein Research, Osaka Univ., Japan
1999-2003: Associate Professor, Institute for Protein Research, Osaka Univ., Japan
1995-1999: Associate Professor, Graduate School of Science, Hokkaido Univ., Japan
1994-1995: Visiting Scientist, Laboratory of Molecular Biology, MRC, UK
1986-1995: Assistant Professor, Photon Factory, KEK, Japan

SPring-8 BL44XU,
A Beamline for Large Biological Macromolecular Assemblies
Eiki Yamashitaa, Keisuke Sakuraia, Masato Yoshimurab, Atsushi NakagawaaaInstitute for Protein Research, Osaka University, Japanb
Taiwan NSRRC, Taiwan Beamline Office at SPring-8, JapanThe Institute for Protein Research (IPR), Osaka University, manages access to a range of highperformance equipment for structural biology, such as the “Synchrotron Beamline at SPring-8”,
the “High Magnetic Field Nuclear Magnetic Resonance (NMR) spectrometers” and the “Advanced
Cryo-Electron Microscopy Suite”. All of these facilities are open to domestic and international
protein science researchers to help support their research.
The beamline BL44XU, named the beamline for macromolecular assemblies, at SPring-8 ismanaged by the IPR since 1999. This beamline is designed for high-precision diffraction datameasurement from large biological macromolecular assemblies. X-ray diffraction from a crystal of
biological macromolecular assembly is generally weak and closely spaced because of its large unit
cell. Therefore, high brilliance and paralleled synchrotron radiation and high-performance detector
with large area are required for diffraction data collection. The light source of this beamline is a
SPring-8 standard type in-vacuum undulator, and X-ray is monochromatized by a liquid-nitrogencooling Si double-crystal monochromator and focused (or collimated) by Rhodium-coatedhorizontal and vertical mirrors. A large, high-efficiency, two-dimensional X-ray photon counter,
EIGER X 16M (DECTRIS Ltd.), is equipped. A SMARGON (SmarAct GmbH), which simulates
4-circle goniometer geometry, achieves the maximal reduction of the blind region in reciprocal
space. The beamline operation software BSS (Beamline Scheduling Software) and a twin-headed
sample auto-changer SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger) are installed
to unify user operation throughout protein crystallography beamlines in the SPring-8. Remote
access and mail-in data collection can be accepted.
Reference[1] Yamashita, E., Nakagawa, A., Biophys. Rev., 11(4), 521-523 (2019).

1

Naoki SAKAI
Senior scientist
Structural Biology Division
Japan Synchrotron Radiation Research Institute (JASRI)
E-mail: nsakai@spring8.or.jp
BIOGRAPHY:
2023-present: Senior scientist, Japan Synchrotron Radiation Research Institute, Japan2022-2023: Research scientist, Japan Synchrotron Radiation Research Institute, Japan
2018-2022: Research scientist, RIKEN SPring-8 Center, Japan
2015-2018: Research scientist, RIKEN. Center for Life Science Technologies, Japan
2011-2014: Research scientist, University of Lübeck, Germany
2006-2010: Assistant Professor, Hokkaido University, Japan
2003-2006: Postdoc, Hokkaido University, Japan

Compound screening pipeline at SPring-8 macromolecular crystallography
beamlines for drug development

Naoki Sakaia,b, Hideo Okumuraa, Hironori Murakamia, Nobuhiro Mizunoa, Yuki Nakamuraa, Go Uenoa,
Hiroaki Matsuurab, Kunio Hiratab, Takuya Masunagaa, Marcel Bokhovea, Takashi Kawamuraa, Seiki
Babaa, Kazuya Hasegawaa, Masaki Yamamotob and Takashi KumasakaaaStructural Biology Division, Japan Synchrotron Radiation Research InstitutebLife Science Research Infrastructure Group, RIKEN SPring-8 Center

SPring-8 operates four macromolecular crystallography beamlines (MX-BLs), BL32XU,
BL41XU, BL45XU, and BL26B1, for public use. Among these, undulator XU beamlimes offer
automated measurements using the data collection system, ZOO [1], eliminating the need forphysical presence. This system enables high-throughput data collection, with over 200 diffraction
datasets obtainable within 24 hours. Consequently, pharmaceutical companies and CROs in Japanand overseas routinely collect substantial data at SPring-8 today.
In conjunction with the high-throughput MX-BLs, we are developing a facility for compound
screening in drug discovery. We have incorporated the acoustic liquid handler Echo to introduce
fragment compounds directly into crystallization drops for crystal soaking. We utilized an in situ
plate diffraction measurement system to get rid of human intervention in cryoprotection during
crystallographic fragment screening. Installed at BL26B1, SPring-8, this system can acquire smallwedge data, and the complete necessary data for structural analysis can be obtained by merging
each data. Our feasibility study demonstrated that the acoustic liquid handler can effectively screen
compounds at room temperature by directly injecting them into drops on the crystallization plateand collecting small-wedge data [2]. Furthermore, we are now designing and examining a new
crystallization plate optimizing for the plate diffraction measurement to enhance its efficiency.
Reference[1] Hirata, K. et al., Acta Cryst. D75, 138–150 (2019).
[2] Okumura, H., Sakai, N. et al., Acta Cryst. F78, 241–251 (2022).
1

Su-Ying Wu
Investigator
National Health Research Institutes
Institute of Biotechnology and Pharmaceutical ResearchE-mail: suying@nhri.edu.tw
BIOGRAPHY:
2011 – present: Investigator, National Health Research Institutes, Taiwan
2006 – 2011: Associate Investigator, National Health Research Institutes, Taiwan
2002 – 2006: Assistant Investigator, National Health Research Institutes, Taiwan

Anti-cancer drug discovery facilitated by x-ray protein crystallography

Tsung-Sheng Wu a , Wen-Hsing Lin a, Hui-Jen Tsai b, Ching-Cheng Hsueha, Tsu Hsua, Pei-Chen Wanga, HuiYou Linb, Yi-Hui Penga, Cheng-Tai Lua, Lung-Chun Leea, Chih-Hsiang Tua, Fang-Chun Kunga, Hui-Yi
Shiaoa, Teng-Kuang Yeha, Jen-Shin Songa, Jia-Yu Changa, Yu-Chieh Sua, Li-Tzong Chen b, Chiung-Tong
Chen a, Weir-Torn Jiaang a, * and Su-Ying Wu a *aInstitute of Biotechnology and Pharmaceutical Research, National Health Research InstitutesbNational Institute of Cancer Research, National Health Research Institutes

In this presentation, the utilization of x-ray protein crystallography within discovery programs at
IBPR, NHRI to guide the lead optimization and elucidate the mode of action of drug candidates
will be discussed. We will highlight the discovery and development of c-KIT inhibitors for thetreatment of gastrointestinal stromal tumors (GISTs). The proto-oncogenic protein c-KIT belongs
to class III receptor tyrosine kinases, and its aberrant activation, resulting from either natural
mutations or drug-induced alterations, is a significant factor in the etiology and drug resistanceobserved in GISTs. Through collaborative efforts across multiple disciplines, we have identified
5-phenyl-thiazol-2-ylamine compounds as potent inhibitors capable of targeting both unactivatedand activated forms of c-KIT.
Notably, these compounds induce conformational rearrangements within key structural motifs of
c-KIT, including the DFG motif, αC-helix, juxtamembrane domain, and activation loop.
Structural biology studies have revealed how these compounds inhibit activated c-KIT, inducing aseries of conformational changes that lead to its transition into an inactive state. Moreover, thesecompounds can effectively inhibit various c-KIT mutants and the proliferation of several GIST celllines. Their favorable pharmacokinetic profiles and efficacy in xenograft models highlight theirpotential as therapeutic agents. Currently, our lead candidate from the c-KIT drug project isadvancing through Phase I clinical trials.

1Hyoun Sook KIM
Senior Scientist
Targeted Therapy Branch, Research InstituteNational Cancer Center
E-mail: hskim@ncc.re.kr
BIOGRAPHY:
2019-present: Senior Scientist, Research Institute, National Cancer Center, Republic of Korea
2016-2019: Assistant Scientist, Research Institute, National Cancer Center, Republic of Korea
2015-2016: Research Professor, Research Institute of Pharmaceutical Sciences, Seoul National
University, Republic of Korea
2012-2015: Research Fellow and Senior Researcher, College of Pharmacy, Seoul National
University, Republic of Korea

Structural Basis for Rab1 Glucosylation by Legionella SetA
on the Early Legionella-Containing Vacuole
Ha Na Ima, Yeon Leea, Dong Man Janga, and Hyoun Sook Kimaa
Research Institute, National Cancer Center, Republic of Korea

Legionnaires’ disease, a severe form of pneumonia primarily targeting the elderly andimmunocompromised, is on the rise in the industrialized world due to Legionella pneumophila.
This bacterium has honed its virulence through long-term coevolution with aquatic protozoa,
enabling it to infect a wide range of phagocytic eukaryotes, including humans. Central to its
pathogenicity is the establishment of the Legionella-containing vacuole (LCV) within host cells,
facilitated by the Dot/Icm type IV secretion system, which delivers a suite of effector proteins tomanipulate host cellular processes. Among these, Legionella glucosyltransferases, specifically
SetA, have emerged as pivotal in altering host cell functions through the modification of key
signaling molecules like Rab1 via glycosylation. This study delves into the structural and molecularbasis of Rab1 glycosylation by SetA, examining the enzyme's interaction with various ligands,
including phosphatidylinositol-3-phosphate (PI3P), crucial for early LCV formation. Through a
combination of crystallography, biochemical assays, biophysical techniques, in silico docking, andSAXS studies, we provide a comprehensive view of SetA’s action mechanism, highlighting its
specificity for GDP-bound Rab1 and PI3P. These insights shed light on Legionella’s intricate
evasion of host immune responses, offering a foundation for future therapeutic strategies against
Legionnaires’ disease.
(unpublished)

1

Chin-Yuan Chang
Associate Professor
Department of Biological Science and Technology
National Yang Ming Chiao Tung University
E-mail: cycytl@nycu.edu.tw

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BIOGRAPHY:
2022-present: Associate Professor, Department of Biological Science and Technology, National
Yang Ming Chiao Tung University, Taiwan
2018-2022: Assistant Professor, Department of Biological Science and Technology, National
Yang Ming Chiao Tung University, Taiwan
2015-2018: Department of Chemistry, Scripps, USA
2011-2015: Genomics Research Center, Academia Sinica, Taiwan

Discovery and Characterization of Genes Conferring Resistance to
Antituberculosis Antibiotic Capreomycina

Shu-Ing Toha, Johan Elaine Keishaa, Yi-Chi Pana and Chin-Yuan Changa
Department of Biological Science and Technology, National Yang Ming Chiao Tung University,
Hsinchu, Taiwan

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from
Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been
identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely
engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA
and IIA, by phosphorylation. In this study, we report that (1) Escherichia coli harboring the cph
gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a newclade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akinto the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA andIIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donortransferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin
phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics
through phosphorylation. Furthermore, Sequence Similarity Network analysis classified 353 Cph
homologues into five major clusters. We examined the function and targeted antibiotics of threeputative resistance proteins in cluster I via bioinformatics, biochemical, and protein structural
analysis. We found that the proteins in cluster I are from various species and indeed confer
resistance to CMN. This study contributes towards understanding the sequence-structure-function
relationships of the phosphorylation resistance genes that confer resistance to CMN.
Reference[1] Toh, S. I., Elaine, K. J., Wang, Y. L., Pan, Y. C., Jhu, Y. H., Hsiao, P. Y., Liao, W. T., Chen,
P. Y., Ko, T. M., Chang, C. Y. Communications Biology, 6, 1282 (2023).
[2] Pan, Y. C., Wang, Y. L., Toh, S. I., Hsu, N. S., Lin, K. H., Xu, Z., Huang, S. C., Wu, T. K., Li,
T. L., Chang, C. Y. ACS Chemical Biology, 17, 138-146 (2022).

1

Ｎien-Jen Hu
Associated Professor
Graduate Institute of Biochemistry
National Chung Hsing University
E-mail: njhu@nchu.edu.tw
BIOGRAPHY:
2020-present: Associated Professor, Graduate Institute for Biochemistry, NCHU, Taiwan
2013-2020: Assistant Professor, Graduate Institute for Biochemistry, NCHU, Taiwan
2010-2013: Postdoctoral Researcher, Diamond Light Synchrotron, UK
2007-2010: Postdoctoral Researcher, Imperial College London, UK

Cryo-EM Structures of KtrAB Reveal the Synergism of ATP and Na+ in
Activating the Bacterial K+ Uptake System
Wesley Tien Chianga, Yao-Kai Changb, Chun-Jung Chenc, Ming-Daw Tsaib and Nien-Jen HuaaGraduate Institute for Biochemistry, NCHU, TaiwanbInstitute of Biochemistry, Academia Sinica, TaiwancNational Synchrotron Radiation Research Center, Taiwan

The high affinity K+ uptake system KtrAB is essential for bacterial survival in low K+ environments.
The activity of KtrAB is regulated by nucleotides and Na+. Previous studies proposed a putative
gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na+
activates KtrAB is still under debate, and the Na+ binding site remains unknown for KtrAB. Here
we present structural and functional results that uncover the Na+ binding site, and discover thesynergism between ATP and Na+ in the activation of KtrAB. The cryo-EM structures of ATP- and
ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) were solved at 2.82 Å and 2.83 Å,
respectively. A cryo-EM density at the intra-dimer interface of ATP-BsKtrA, assigned to Ca2+ orMg2+ previously, was observed even in the presence of divalent cation chelators. Na+ was assigned
to the site, as supported by X-ray crystallography and ICP-MS. Thermostability assays andfunctional studies demonstrated that Na+ binding stabilizes ATP-bound BsKtrAB complex andenhances the K+ flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that
BsKtrB Arg417 and Phe91 serve as a gate. The gate is closed at the ADP-bound state, and open
when BsKtrB D1M2b helix forms a hairpin at the ATP-bound state. The results are likely
applicable to other Na+-activated K+ channels.
Reference[1] Wesley T. Chiang, et al. Nature Communications, 2024, Accepted.
[2] Vieira Pires et. al., 2013, Nature, 496(7445).
1

Sachiko TOMA-FUKAI
Associate Professor
Division of Material Science
Graduate School of Science and Technology
NARA Institute of Science and Technology
E-mail: toma@ms.naist.jp

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BIOGRAPHY:
2018–Present: Associate Professor, Nara Institute of Science and Technology, Japan
2006-2018: Assistant Professor, Graduate School of Pharmaceutical Sciences, The Univ. of Tokyo
2003–2006: Assistant Professor, Graduate School of Pharmaceutical Sciences, Kumamoto Univ.,
Japan

Structural study of the nuclear transportation mechanism of PPARg
by Transportin-1
Sachiko Toma-Fukai a, b, Yutaro Nakamurab, Akihiro Kawamotoc, Hikaru Shimizub, Koki Hayamaa,
Mika Hirosec, and Takayuki Katoc, Hironari Kamikubo a, and Toshiyuki ShimizubaGraduate School of Science and Technology, Nara Institute of Science and Technology,b
Graduate School of Pharmaceutical Sciences, The University of TokyocInstitute for Protein Research, Osaka University

The spatial and temporal control of protein is essential for normal cellular function. Proteinsworking in the nucleus have nuclear localization signal (NLS) sequences and are escorted bycognate nuclear transport receptors. A wealth of experimental data about NLS has been
accumulated, and nuclear transportation mechanisms have been established.
The peroxisome proliferator-activated receptors (PPARs) are a member of the nuclear receptor
family proteins considered an integral part of essential human drug target families (GPCR, ion
channels, protein kinase, and nuclear receptor). PPARs control cellular processes, including lipid
regulation and carbohydrate metabolism. PPARg, the best-studied member of the PPAR family, isexpressed in white and brown adipocytes and regulates adipocyte differentiation, lipid storage, andrelease. PPARg is the master regulator of fat-cell formation.
Recently, our group reported that Transportin-1, also known as karyopherin-b2, works as thenuclear transportation receptor of PPARg [1]. Trn1 directly recognizes the PY-NLS sequence andthe RGG regions of the cargo proteins. However, PPARg lacks the typical NLS and its recognition
mechanism remains largely unknown.
We determined the Cryo-EM structure of PPARg in complex with Transportin-1. Transportin1 gripped the folded DNA binding domain and the Hinge region of PPARg, indicating that PPARg
is recognized a folded domain with an extended region as an NLS, not a canonical unstructured
signal sequence, confirmed by the mutation analyses in vitro and cultured cells. Our study is thefirst snapshot structure working in nuclear transportation, not in transcription, of PPARg.
Reference[1] T. Teratani, et al, Free radical biology & medicine, 156, 45-56 (2020).

1

Hui-Chun Cheng
Associate Professor
Institute of Bioinformatics and Structural Biology
National Tsing Hua University
E-mail: hccheng@life.nthu.edu.tw
BIOGRAPHY:
2022-present: Associate Professor, Institute of Bioinformatics and Structural Biology, NTHU,
Taiwan2014-2022: Assistant Professor, Institute of Bioinformatics and Structural Biology, NTHU, Taiwan

Molecular Basis of Cep57 in Centrosome Regulation
Hui-Chun Cheng (鄭惠春)*, Hung-Wei Yeh (葉弘瑋), Po-Pang Chen (陳博邦)
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Taiwan

Abstract
As a coiled-coil scaffold at the pericentriolar matrix (PCM), human Cep57 controls centrosome
biogenesis for faithful cell division[1]. Truncation mutations of Cep57 are genetically linked to themosaic-variegated aneuploidy (MVA) syndrome[2]. In interphase, Cep57 forms a complex with
Cep63 and Cep152, critical factors for the centrosome cycle[3]. However, the molecular basis of
Cep57 and its functional interplay with the other essential scaffolding proteins for centrosome
integrity remains unclear. Through the integration of multiple structural and biophysical methods,
we illustrate that Cep57 undergoes liquid-liquid phase separation (LLPS) mediated by threeessential domains: coiled-coil NTD, polybasic LMN, and CTD. Functionally, Cep57 condensate
promotes microtubule nucleation via the LMN-mediated tubulin concentration. Cep63 regulates
the temporal control of the centrosome by controlling Cep57 assembly, expansion, and microtubule
polymerization activity. Overexpressing LLPS-blocking Cep57 constructs, including an MVA
truncation mutation, resulted in centrosome over-duplication. Our mechanistic study reveals that
Cep57 LLPS regulates human centrosome[4].
Reference[1] Watanabe, K., Takao, D., Ito, K. K., Takahashi, M. & Kitagawa, D. The Cep57-pericentrin
module organizes PCM expansion and centriole engagement. Nat. Commun. 2019, 10, 931.
[2] Aziz, K. et al. Mosaic-variegated aneuploidy syndrome mutation or haploinsufficiency inCep57 impairs tumor suppression. J. Clin. Invest. 2018, 128, 3517–3534.
[3] Lukinavičius, G. et al. Selective chemical crosslinking reveals a Cep57-Cep63-Cep152
centrosomal complex. Curr. Biol. 2013, 23, 265–270.
[4] Yeh, H.-W. et al. Cep57 Regulates Human Centrosomes through Multivalent Interactions.
PNAS, under revision1

Terukazu NOGIAssociate Professor
Graduate School of Medical Life Science
Yokohama City University
E-mail: nogi@yokohama-cu.ac.jp

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BIOGRAPHY:
2011-present: Associate Professor, Yokohama City University, Japan
2007-2011: Assistant Professor, Institute for Protein Research, Osaka University, Japan
2004-2007: Research Associate, Institute for Protein Research, Osaka University, Japan
2002-2004: Postdoctoral Research Fellow, Max-Planck-Institute of Biophysics, Germany
2001-2002: Postdoctoral Research Fellow, Photon Factory, KEK, Japan

Structural basis for intramembrane proteolysis by bacterial site-2 proteases
Nogi, T.
Graduate School of Medical Life Science, Yokohama City University

Intramembrane proteases (IMPs) catalyze a hydrolysis within the lipid bilayer, a cellular processtermed as intramembrane proteolysis. The IMPs are thought to maintain membrane proteostasis byremoving unnecessary and potentially cytotoxic membrane proteins. The IMPs are also known toperform signal transduction by cleaving membrane-associated signaling molecules. For instance,
metalloprotease-type site-2 protease (S2P) cleaves membrane-bound transcription factor
precursors to regulate lipid metabolism and unfolded protein response in human. E. coli also
possesses an S2P homologue, RseP, which activates transcription factors regulating
extracytoplasmic stress response and iron uptake. Additionally, RseP is known to remove remnant
signal peptides released from secretory proteins while cleaving an endogenous toxin HokB, which
induces persister formation, and suppressing cytotoxicity [1]. Although the physiological functionsof RseP have been extensively explored as mentioned above, the molecular mechanism underlying
the substrate selection and cleavage remains unclear. In fact, the substrates identified so far show
no apparent consensus on the primary structure whereas the cleavage is not promiscuous. We
therefore have been working on structural analysis and have determined crystal structures of E. coli
RseP and its orthologue in complex with an inhibitor [2]. Structure-based biochemical analysis
suggests that RseP undergoes conformational changes during the substrate accommodation as theactive site is surrounded by transmembrane helices and peripheral membrane-associated domains.
Substrate membrane proteins are likely to be unwound into an extended conformation throughinteraction with the membrane-embedded β-sheet and to be clamped by a conserved asparagine
located close to the active site. We will discuss the cleavage mechanism in more detail by alsopresenting our recent results.
Reference[1] Yokoyama, T. et al., mBio, 14 (4), e0108623 (2023).
[2] Imaizumi, Y. et al., Sci Adv, 8 (34), eabp9011 (2022).

1

Genji KURISU
Professor
Institute for Protein Research
Osaka University
E-mail: gkurisu@protein.osaka-u.ac.jp
BIOGRAPHY:
2024-present: Director, Institute for Protein Research, Osaka Univ., Japan2017-present: Head, Protein Data Bank Japan (PDBj), Osaka Univ., Japan
2009-present: Professor, Institute for Protein Research, Osaka Univ., Japan
2004-2009: Associate Professor, Department of Life Sciences, Univ. Tokyo, Japan
2002-2003: Visiting Scientist, Department of Biological Sciences, Purdue Univ., USA
1997-2004: Assistant Professor, Institute for Protein Research, Osaka Univ., Japan

Structure of cyanobacterial photosystem I complexed with ferredoxin andcyrtochrome c6 at 1.97 Å resolutionaGenji Kurisua,bLaboratory of Protein Crystallography, Institute for Protein Research, Osaka Universityb
Laboratory of Protein Databases, Institute for Protein Research, Osaka University

Cryo-EM structure of the trimeric Photosystem I (PSI) from thermophilic cyanobacterium,
Thermosynechococcus elongatus BP-1, complexed with its two electron transfer partner proteinsof Cytochrome c6 (Cyt c6) and Ferredoxin (Fd), has been solved to the highest record of 1.97 Å
resolution [1], thus revealing the specific molecular interaction of its donor and acceptor proteins.
Each protomer contains newly assigned seventeen lipids, three pigments, additional forty residues,and six water molecules at the interface with Fd. Fd-binding to PSI was thermodynamicallycharacterized also by Isothermal Titration Calorimetry measurement, which was complementary
to and consistent with cryo-EM analysis. The cryo-EM densities for Cyt c6 in our study are the firstexperimental result for visualization of Cyt c6 bound to PSI, even though the densities themselves
were not so intense, which might be induced by productive binding of Fd on the opposite side of
PSI. Based on all our new findings, a safe and productive electron transfer mechanism
accompanying two-way trans-membrane communications within the cyanobacterial PSI was
proposed.
Reference[1] Li, J., Hamaoka, N., Makino, F., Kawamoto, A., Lin, Y., Rögner, M., Nowaczyk, M.M., Lee,
Y.-H., Namba, K., Gerle, C., Kurisu, G, Commun Biol, 5 (1), 951 (2022)1Danaya PAKOTIPRAPHA
Associate Professor
Department of Biochemistry &Center for Excellence in Protein and Enzyme Technology
Faculty of Science, Mahidol University
E-mail: danaya.pak@mahidol.ac.th

Photo

BIOGRAPHY:
2020–present: Associate Professor, Faculty of Science, Mahidol University, Thailand
2015–2020: Assistant Professor, Faculty of Science, Mahidol University, Thailand
2012–2015: Lecturer, Faculty of Science, Mahidol University, Thailand

Versatility of MarR domain in the regulation of phenolic compound utilization
Permkun Permsirivisarn a, Anan Yuenyao a, Nopphon Petchyam a, Nuntaporn Kamonsutthipaijitb,
Ratana Charoenwattanasatienb, Nuttawan Pramanpolb, Wipa Sugintac, Pimchai Chaiyenc,
and Danaya PakotipraphaaaDepartment of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of
Science, Mahidol University, Thailand.
bSynchrotron Light Research Institute (Public Organization), Nakhon Ratchasima, Thailand.
c
SynSchool of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science andTechnology (VISTEC), Rayong 21210, Thailand.

Some bacteria are capable of converting the phenolic compound p-hydroxyphenylacetate(HPA) to pyruvate and succinate for cellular metabolism. The genes that encode the enzymes
required for such conversion are found in a gene cluster that is regulated by a transcription regulator
in the MarR family, HpaR. Biochemical and structural characterization of Acinetobacterbaumannii HpaR (AbHpaR) and its complex with cognate DNA revealed that AbHpaR binds
upstream of the divergently transcribed hpaA gene and the meta-cleavage operon, as well as the
hpaR gene, thereby repressing their transcriptions by blocking access of RNA polymerase. DNA
binding specificity of AbHpaR is achieved by a combination of both direct and indirect DNA
sequence readouts. The presence of 3,4-dihydroxyphenylacetate (DHPA), which is the substrateof the meta-cleavage reactions, weakens DNA binding by AbHpaR and likely leads to expressionof the target genes [1].
In addition to being found in HpaR, the MarR domain is also found in the enzyme phydroxyphenylacetate 3-hydroxylase (HPAH), which catalyzes the first step in the catabolism of
HPA. Here, the MarR domain inhibits the flavin reductase activity in the absence of aromatic
substrates. X-ray crystallographic and small-angle X-ray scattering (SAXS) studies revealed that
HPAH undergoes a substantial conformational change in the presence of HPA, concomitant with
the increase in the rate of flavin reduction [2].
References[1] Permsirivisarn, P. et al. FEBS J. 289, 3217–3240 (2022).
[2] Yuenyao, A. et al. Arch. Biochem. Biophys. 653, 24–38 (2018).
1

Shih-Ming Lin
Assistant Professor
Department of Biotechnology and Bioindustry Sciences
National Cheng Kung University
E-mail: smlin@mail.ncku.edu.tw
BIOGRAPHY:
2016- present: Assistant Professor, Department of Biotechnology and Bioindustry Sciences,
NCKU, Taiwan

Deciphering the Calcium-Dependent Activation Mechanism of Vibrio
campbellii α-Hemolysin

Hsiang Changa, Yu-Chuan Chiua, Min-Chi Yehb, Chun-Hsiung Wangb, Yu-An Chena, Meng-Chiao Hob &Shih-Ming Linaa
Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan,
TaiwanbInstitute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

Vibrio α-hemolysins, as essential β-pore-forming toxins, play a crucial role in the pathogenesisof bacterial infections by facilitating host cell disruption through membrane lysis. These toxins
undergo a proteolytic maturation process to transform from inactive precursors into their active
forms. Our study has identified another critical factor modulating the hemolytic activation of Vibrio
campbellii α-hemolysin (VcαHL), emphasizing the significant role of calcium ions in toxin
stimulation. We demonstrate that calcium ions not only enhance the hemolytic activity of VcαHLbut are also crucial for its oligomerization on cell membranes. Utilizing X-ray crystallography andcryo-electron microscopy, we revealed the structural transitions of VcαHL from its proenzyme
state to active oligomerization, highlighting the conformational changes essential for its functional
assembly. These high-resolution structures and mutagenesis studies revealed a calcium-binding
module within VcαHL that auto-inhibits the toxin assembly until calcium ions bind. Additionally,another set of calcium-binding sites were identified in the β-prism lectin domain of VcαHL,suggesting that calcium binding may also play a role in the membrane association of this hemolytic
toxin. These findings deepen our understanding of VcαHL's functional mechanism and open
avenues for developing targeted therapeutic strategies against Vibrio infections.

Reference[1] Yu-Chuan Chiu, Min-Chi Yeh, Chun-Hsiung Wang, Yu-An Chen, Hsiang Chang, Han-You
Lin, Meng-Chiao Ho, & Shih-Ming Lin* Structural basis for calcium-stimulating pore formationof Vibrio α-hemolysin. Nature Communications, 14, 5946. (2023)

1D260, 2nd floor, Activity Center
National Synchrotron Radiation Research Center
Hsinchu, Taiwan