Researcher basic information

• Ph.D., University of Liverpool, Mar. 2020

• https://researchmap.jp/chai_gopalasingam?lang=en

• https://orcid.org/0000-0002-9014-9380
• https://jglobal.jst.go.jp/detail?JGLOBAL_ID=202201015400814340

• 40876257

• https://orcid.org/0000-0002-9014-9380

• 202201015400814340

• Denitrification Enzymes
• Respiratory Complexes
• X-ray Crystallography
• Cryogenic Electron Microscopy (Single Particle Analysis)
• Metalloproteins
• Membrane Protein Structural Biology

• Life Science, Biophysics
• Life Science, Functional biochemistry
• Life Science, Structural biochemistry, Structural Biology

• Bachelor's degree program, School of Science
• Master's degree program, Graduate School of Chemical Sciences and Engineering
• Doctoral (PhD) degree program, Graduate School of Chemical Sciences and Engineering

Research activity information

• Sep. 2021, Integrated Biometal Science, Young Researcher Poster Award
  Exploring native structures of nitric oxide reductase using cryoEM
• May 2019, Instruct-ERIC, Student Bursary Award
• Jul. 2018, PDBe (Protein Databank in Europe), UKSR50 Poster Award
• Sep. 2017, RIKEN, RIKEN Summer School Biology Poster Award

• Structural basis of Neisseria meningitidis quinol dependent nitric oxide reductase activation by dimerization
  Chai C. Gopalasingam; Haruka Egami; Hideki Shigematsu; Masatora Sakaue; Kouki Fukumoto; Christoph Gerle; Masaki Yamamoto; Yoshitsugu Shiro; Kazumasa Muramoto; Takehiko Tosha
  Communications Biology, 27 Mar. 2026
  Scientific journal
• Substrate specificity and action mechanism of the HerA-NurA nuclease from the hyperthermophilic archaeon Thermococcus kodakarensis
  Keishiro Uda; Takeshi Yamagami; Sonoko Ishino; Christoph Gerle; Chai C. Gopalasingam; Hideki Shigematsu; Tomoyuki Numata; Yoshizumi Ishino
  mBio, 11 Mar. 2026
  Scientific journal
• Sequential structural rearrangements at the PAM-distal site of a type I-F3 CRISPR-Cas effector enabling RNA-guided DNA transposition
  Kazuki Ishihara; Shunsuke Matsumoto; Christoph Gerle; Chai C Gopalasingam; Hideki Shigematsu; Tsuyoshi Shirai; Tomoyuki Numata
  Nucleic Acids Research, 54, 1, Oxford University Press (OUP), 05 Jan. 2026
  Scientific journal, Abstract
  
  Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA-guided DNA binding and interactions with transposition-associated proteins. Although efficient sequence-specific DNA integration requires both precise target DNA recognition and coordinated interactions between effectors and transposition-associated proteins, the underlying mechanism remains elusive. Here, we determined three cryo-EM structures of target DNA-bound type I-F3 TniQ-Cascade from Vibrio parahaemolyticus, revealing how Cas8/5 recognizes the protospacer adjacent motif (PAM) and identifying a key residue responsible for the cytidine preference at position -2 of the PAM. We revealed mismatch tolerance at the PAM-proximal site. Structural analyses showed that correct base pairing at the PAM-distal site correlates with conformational changes in the Cas8/5 helical bundle and TniQ, bending the DNA to guide its downstream region toward the transposition machinery. Together, these dynamic rearrangements at the PAM-distal region provide insights into the licensing mechanism of type I-F3 CAST transposition and highlight its potential for genome engineering applications.
• Design, synthesis, and structural analysis of an inhibitor of the gastric proton pump with a diaza-tricyclic skeleton
  Nariyoshi Umekubo; Airi Hashizume; Haruki Saito; Satoru Kato; Chisato Kanai; Chai C. Gopalasingam; Christoph Gerle; Hideki Shigematsu; Atsushi Yoshimori; Kazuhiro Abe; Satoshi Yokoshima
  Organic & Biomolecular Chemistry, 2026
  Scientific journal
• Cryo-EM structure of the ATP11C Q79E mutant reveals the structural basis for altered Phospholipid recognition
  Yuheng Qian; Chai C. Gopalasingam; Christoph Gerle; Hideki Shigematsu; Kazuhiro Abe; Atsunori Oshima
  Journal of Biological Chemistry, Jan. 2026
  Scientific journal
• A unique gating mechanism revealed by the cryo-EM structure of monomeric ATP9A flippase
  Kazuhiro Abe; Parthiban Marimuthu; Yuheng Qian; Chai C. Gopalasingam; Christoph Gerle; Hideki Shigematsu; Kotaro Tanaka; Himanshu Khandelia
  Journal of Biological Chemistry, Oct. 2025
  Scientific journal
• Molecular Structure of the Na+,K+-ATPase α4β1 Isoform in Its Ouabain-Bound Conformation
  Kazuhiro Abe; Jeff McDermott; Hridya Valia Madapally; Parthiban Marimuthu; Chai C. Gopalasingam; Christoph Gerle; Hideki Shigematsu; Himanshu Khandelia; Gustavo Blanco
  International Journal of Molecular Sciences, 19 Nov. 2024
  Scientific journal
• Corrigendum to "Human F-ATP synthase as a drug target" [Pharmacol. Res. 209 (2024) 107423].
  Christoph Gerle; Chimari Jiko; Atsuki Nakano; Ken Yokoyama; Chai C Gopalasingam; Hideki Shigematsu; Kazuhiro Abe
  Pharmacological research, 209, 107467, 107467, Nov. 2024, [International Magazine]
  English
• Human F-ATP synthase as a drug target
  Christoph Gerle; Chimari Jiko; Atsuki Nakano; Ken Yokoyama; Chai C. Gopalasingam; Hideki Shigematsu; Kazuhiro Abe
  Pharmacological Research, 209, 107423, 107423, Elsevier BV, Nov. 2024
  Scientific journal
• NDT-C11 as a viable novel detergent for single particle cryo-EM.
  Chimari Jiko; Jiannan Li; Youngsun Moon; Yoshito Tanaka; Chai C Gopalasingam; Hideki Shigematsu; Pil Seok Chae; Genji Kurisu; Christoph Gerle
  ChemPlusChem, e202400242, 17 Jun. 2024, [International Magazine]
  English, Scientific journal, Single particle cryo electron microscopy (cryo-EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo-grid vitrification is crucial. Although small molecule amphiphiles - detergents - are the most widely used membrane mimetic, specific tailoring of detergent structure for single particle cryo-EM is rare and the demand for effective detergents not satisfied. Here, we compare the popular detergent lauryl maltose-neopentyl glycol (LMNG) with the novel detergent neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free detergent and when bound to two types of multisubunit membrane protein complexes - cyanobacterial photosystem I (PSI) and mammalian F-ATP synthase. We conclude that NDT-C11 has high potential to become a very useful detergent for single particle cryo-EM of integral membrane proteins.
• Monomer-dimer structural comparison in quinol-dependent nitric oxide reductase reveals a functional basis for superior enzymatic activity in the dimer
  Chai C. Gopalasingam; Haruka Egami; Hideki Shigematsu; Masatora Sakaue; Kouki Fukumoto; Christoph Gerle; Masaki Yamamoto; Yoshitsugu Shiro; Kazumasa Muramoto; Takehiko Tosha
  17 May 2024
• Structural insights into thermophilic chaperonin complexes
  Zengwei Liao; Chai C. Gopalasingam; Masafumi Kameya; Christoph Gerle; Hideki Shigematsu; Masaharu Ishii; Takatoshi Arakawa; Shinya Fushinobu
  Structure, Elsevier BV, Mar. 2024
  Scientific journal
• Deep learning driven de novo drug design based on gastric proton pump structures
  Kazuhiro Abe; Mami Ozako; Miki Inukai; Yoe Matsuyuki; Shinnosuke Kitayama; Chisato Kanai; Chiaki Nagai; Chai C. Gopalasingam; Christoph Gerle; Hideki Shigematsu; Nariyoshi Umekubo; Satoshi Yokoshima; Atsushi Yoshimori
  Communications Biology, 6, 1, Springer Science and Business Media LLC, 19 Sep. 2023
  Scientific journal, Abstract
  
  Existing drugs often suffer in their effectiveness due to detrimental side effects, low binding affinity or pharmacokinetic problems. This may be overcome by the development of distinct compounds. Here, we exploit the rich structural basis of drug-bound gastric proton pump to develop compounds with strong inhibitory potency, employing a combinatorial approach utilizing deep generative models for de novo drug design with organic synthesis and cryo-EM structural analysis. Candidate compounds that satisfy pharmacophores defined in the drug-bound proton pump structures, were designed in silico utilizing our deep generative models, a workflow termed Deep Quartet. Several candidates were synthesized and screened according to their inhibition potencies in vitro, and their binding poses were in turn identified by cryo-EM. Structures reaching up to 2.10 Å resolution allowed us to evaluate and re-design compound structures, heralding the most potent compound in this study, DQ-18 (N-methyl-4-((2-(benzyloxy)-5-chlorobenzyl)oxy)benzylamine), which shows a K_i value of 47.6 nM. Further high-resolution cryo-EM analysis at 2.08 Å resolution unambiguously determined the DQ-18 binding pose. Our integrated approach offers a framework for structure-based de novo drug development based on the desired pharmacophores within the protein structure.
• Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases
  Yuya Nishida; Sachiko Yanagisawa; Rikuri Morita; Hideki Shigematsu; Kyoko Shinzawa-Itoh; Hitomi Yuki; Satoshi Ogasawara; Ken Shimuta; Takashi Iwamoto; Chisa Nakabayashi; Waka Matsumura; Hisakazu Kato; Chai Gopalasingam; Takemasa Nagao; Tasneem Qaqorh; Yusuke Takahashi; Satoru Yamazaki; Katsumasa Kamiya; Ryuhei Harada; Nobuhiro Mizuno; Hideyuki Takahashi; Yukihiro Akeda; Makoto Ohnishi; Yoshikazu Ishii; Takashi Kumasaka; Takeshi Murata; Kazumasa Muramoto; Takehiko Tosha; Yoshitsugu Shiro; Teruki Honma; Yasuteru Shigeta; Minoru Kubo; Seiji Takashima; Yasunori Shintani
  Nature Communications, 13, 1, Springer Science and Business Media LLC, 08 Dec. 2022
  Scientific journal, Abstract
  
  Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.
• Frontiers in metalloprotein crystallography and cryogenic electron microscopy
  Chai C. Gopalasingam; S. Samar Hasnain
  Current Opinion in Structural Biology, 75, 102420, 102420, Elsevier {BV}, Aug. 2022
  English, Scientific journal
• Short-lived intermediate in N ₂ O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography
  Takashi Nomura; Tetsunari Kimura; Yusuke Kanematsu; Daichi Yamada; Keitaro Yamashita; Kunio Hirata; Go Ueno; Hironori Murakami; Tamao Hisano; Raika Yamagiwa; Hanae Takeda; Chai Gopalasingam; Ryota Kousaka; Sachiko Yanagisawa; Osami Shoji; Takashi Kumasaka; Masaki Yamamoto; Yu Takano; Hiroshi Sugimoto; Takehiko Tosha; Minoru Kubo; Yoshitsugu Shiro
  Proceedings of the National Academy of Sciences, 118, 21, Proceedings of the National Academy of Sciences, 25 May 2021
  English, Scientific journal, Significance
  
  The short-lived intermediate formed during the reduction of nitric oxide (NO) to nitrous oxide (N
  2
  O) in denitrification, microbial anaerobic respiration, is a key state for understanding the generation mechanism of N
  2
  O, known not only as a greenhouse gas but also as an ozone-depleting substance on the global level. This paper combined state-of-the-art, time-resolved techniques, such as flow-flash infrared spectroscopy and X-ray free electron laser-based crystallography, and captured the intermediate of a P450-type NO reductase at the atomic and electronic levels. The intermediate was identified as a singly protonated Fe
  3+
  –NHO
  •−
  radical, offering insights into a radical–radical coupling mechanism for the N–N bond formation in N
  2
  O generation.
  
• The active form of quinol-dependent nitric oxide reductase from Neisseria meningitidis is a dimer
  M. Arif M. Jamali; Chai C. Gopalasingam; Rachel M. Johnson; Takehiko Tosha; Kazumasa Muramoto; Stephen P. Muench; Svetlana V. Antonyuk; Yoshitsugu Shiro; Samar S. Hasnain
  IUCrJ, 7, 3, 404, 415, International Union of Crystallography ({IUCr}), 01 May 2020
  Scientific journal
• Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo–electron microscopy
  Chai C. Gopalasingam; Rachel M. Johnson; George N. Chiduza; Takehiko Tosha; Masaki Yamamoto; Yoshitsugu Shiro; Svetlana V. Antonyuk; Stephen P. Muench; S. Samar Hasnain
  Science Advances, 5, 8, American Association for the Advancement of Science ({AAAS}), 02 Aug. 2019
  Scientific journal
• Catalytically important damage-free structures of a copper nitrite reductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography
  Thomas P. Halsted; Keitaro Yamashita; Chai C. Gopalasingam; Rajesh T. Shenoy; Kunio Hirata; Hideo Ago; Go Ueno; Matthew P. Blakeley; Robert R. Eady; Svetlana V. Antonyuk; Masaki Yamamoto; S. Samar Hasnain
  IUCrJ, 6, 4, 761, 772, International Union of Crystallography ({IUCr}), 01 Jul. 2019
  Scientific journal, Copper-containing nitrite reductases (CuNiRs) that convert NO₂⁻to NO via a Cu_CAT–His–Cys–Cu_ETproton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that Asp_CATand His_CATare deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D₂O) is positioned with one deuteron directed towards Asp_CAT O^δ1and one towards His_CAT N^∊2. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D₂O molecule as opposed to D₃O⁺or OD⁻, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated Asp_CATand is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with Asp_CAT. Upon the binding of NO₂⁻a proton is transferred from the bridging water to the O^δ2atom of Asp_CAT, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from Asp_CATto the bound nitrite, enabling the reaction to proceed.
• Crystal structure of the TreS:Pep2 complex, initiating α-glucan synthesis in the GlgE pathway of mycobacteria
  Chai Gopalasingam
  Journal of Biological Chemistry, 294, 18, 7348, 7359, Elsevier BV, 03 May 2019
  English, Scientific journal

• Structural basis of enhanced NO reduction via dimerization of membrane-bound nitric oxide reductase
  Chai C. Gopalasingam
  Hokkaido Univeristy- Seoul National University Symposium, 26 Jan. 2026
  26 Jan. 2026 - 27 Jan. 2026, [Invited]
• About the RIKEN SPring-8 Public Use CryoEM Facility
  Chai Gopalasingam
  3rd Biometal Science Summer Camp, 09 Sep. 2023, Oral presentation
  08 Sep. 2023 - 09 Sep. 2023, [Invited]
• Microscopes in the mountains: Progress and Challenges from the RIKEN SPring-8 cryoEM facility
  Chai Gopalasingam
  Astbury Biostructure Laboratory Monthly Cryo EM Seminar, 06 Jun. 2023, Public discourse
  [Invited]
• Exploring the function (and dysfunction) of qNOR by cutting edge cryoEM
  Chai Gopalasingam
  35th Summer Seminar on Bioinorganic Chemistry, 07 Sep. 2023, Invited oral presentation
  07 Sep. 2023 - 2023, [Invited]
• CryoEM Structure of monomeric quinol-dependent nitric oxide reductase reveals structural basis of enhanced nitric oxide detoxification
  Chai Gopalasingam; Haruka Egami; Kouki Fukumoto; Hideki Shigematsu; Masatora Sakaue; Takehiko Tosha; Masaki Yamamoto; Kazumasa Muramoto; Yoshitsugu Shiro
  AsBIC 10, 29 Nov. 2022, Invited oral presentation
  28 Nov. 2022 - 03 Dec. 2022, [Invited]