This article has been reviewed according to Science X's editorial process and policies. Editors have highlighted the following attributes while ensuring the content's credibility: 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).
The paper was published in the International Journal of Molecular Sciences. 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.
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