From a rigid molecular pair to flexible assemblies with moving domains, the enzymes that build sugar chains display strikingly different behaviors. A research team at the Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, working with collaborators at Nagoya City University, Nagoya University, Kyoto University, Osaka University, and the Institute for Molecular Science (IMS), has captured these differences using high-speed atomic force microscopy (HS-AFM). By combining molecular movies with X-ray crystallography and solution biophysics, the team revealed a spectrum of dynamic architectures that static structural models alone cannot fully describe.
Sugar chains, or glycans, contribute to cell-cell communication and the organization of the extracellular matrix. Glycosyltransferases build these chains by transferring sugar units to acceptor molecules. Some contain multiple structural domains that contribute to catalysis or substrate recognition. Crystal structures and cryo-EM reconstructions can reveal different conformations and assembly states, but do not directly track the movements of individual molecules over time. For enzymes that extend glycan chains, domain arrangement and motion may help accommodate the growing chain and coordinate successive reactions. This study compared assembly and motion across several enzymes using a common set of complementary approaches.
The researchers prepared soluble recombinant forms of human POMGNT2 and LARGE1 and the bacterial chondroitin-building enzyme K4CP. They also carried out exploratory analyses of L137, a glycosyltransferase candidate from a giant virus, Mimivirus. Mass photometry assessed the assembly states of all four proteins, while HS-AFM tracked individual particles adsorbed onto a supporting surface in liquid. Additional solution measurements helped establish how the proteins assembled, and a newly determined crystal structure of POMGNT2 provided a reference for interpreting its observed shape.
POMGNT2 formed a stable dimer, a pair of protein molecules, that retained its shape with little large-scale motion. LARGE1 displayed flexible movements between its domains and a range of assembly states associated with different protein concentrations. K4CP showed the most dramatic movements: its domains repeatedly opened and closed, while solution measurements revealed weak, concentration-dependent dimerization. Adding chondroitin oligosaccharides, which serve as acceptor substrates, also produced a qualitative tendency toward more compact K4CP conformations. The viral candidate L137 expanded this picture of structural diversity: it was predominantly monomeric, with three flexibly arranged lobes.
Together, the findings reveal a spectrum of dynamic organizational strategies, from the relatively rigid POMGNT2 dimer to the flexible, reversible assemblies of LARGE1 and K4CP. These contrasting architectures suggest a connection between molecular organization and catalytic demands: a stable framework may support precise substrate recognition, while flexible domains may accommodate an elongating glycan chain. By bringing enzyme motion into view, the study provides a foundation for understanding glycan synthesis through both structure and dynamics. Building on these observations of purified soluble proteins, future studies will connect domain motion with catalytic activity and examine how membrane attachment shapes enzyme behavior.
Hirokazu Yagi
Graduate School of Pharmaceutical Sciences, Nagoya City University
Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences
You-Rong Lin
Exploratory Research Center on Life and Living Systems (ExCELLS) / Institute for Molecular Science (IMS), National Institutes of Natural Sciences
Yui Kanaoka
Department of Physics, Graduate School of Science, Nagoya University
Fumiko Umezawa
Graduate School of Pharmaceutical Sciences, Nagoya City University
Akemi Kim
Graduate School of Pharmaceutical Sciences, Nagoya City University
Kotaro Tomuro
Graduate School of Pharmaceutical Sciences, Nagoya City University
Ken Morishima
Institute for Integrated Radiation and Nuclear Science, Kyoto University
Atsuji Kodama
Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences
Kentaro Ishii
Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences
Department of Biotechnology, Graduate School of Engineering, Osaka University
Susumu Uchiyama
Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences
Department of Biotechnology, Graduate School of Engineering, Osaka University
Tadashi Satoh
Graduate School of Pharmaceutical Sciences, Nagoya City University
Masaaki Sugiyama
Institute for Integrated Radiation and Nuclear Science, Kyoto University
Takayuki Uchihashi
Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences
Department of Physics, Graduate School of Science, Nagoya University
Department of Physics and Institute for Glyco-Core Research (iGCORE), Nagoya University
Quantum-Based Frontier Research Hub for Industry Development (Q-BReD), Nagoya University
Koichi Kato
Exploratory Research Center on Life and Living Systems (ExCELLS) / Institute for Molecular Science (IMS), National Institutes of Natural Sciences
Graduate School of Pharmaceutical Sciences, Nagoya City University
International Journal of Molecular Sciences
Experimental study
Not applicable
Dynamic Organizational Strategies of Multidomain Glycosyltransferases Revealed by High-Speed AFM and Solution Biophysics
19-Jul-2026