A research group from the School of Life Science and Technology at the Institute of Science Tokyo, including Mariko Kojima (currently Assistant Professor at Tohoku University), Graduate Student Xinchen Yao (at the time of research), Assistant Professor Satoshi Abe (currently Associate Professor at Kyoto Prefectural University), Assistant Professor Tadaomi Furuta, and Professor Takafumi Ueno, announced the development of a new analytical platform for elucidating the flexible structure and dynamics of glycans at the atomic level. For the first time at atomic resolution, they determined the binding structure of melezitose, one of the trisaccharides that was previously difficult to analyze using conventional methods. By combining molecular dynamics simulations using a supercomputer, the researchers also revealed how glycan flexibility is controlled by the binding site. Their findings are expected to be applied to drug discovery research and functional analysis of sugar molecules. The results were published in Small Structures on October 23.
Glycans are important molecules involved in various biological phenomena such as cell-cell recognition and signal transduction. However, due to their flexible and complex structures, it has been difficult to understand their three-dimensional structures and movements using conventional analytical methods. In particular, how glycans "fluctuate" while functioning when bound to proteins has been a major challenge in life sciences and drug discovery research.
In this study, the research group focused on the property of galectin-10 (Gal-10), a sugar-binding protein (lectin), when crystallizing spontaneously in vivo. They developed an analytical technique for studying the structure and fluctuations of sugar molecules using these crystals as a scaffold. Gal-10 is a lectin abundantly found in white blood cells and forms "Charcot-Leyden crystals" in diseases such as asthma.
By applying their newly developed "cell-free protein crystallization (CFPC)" to Gal-10, they succeeded in mass-producing high-quality crystals in vitro in just one day. Although Gal-10 crystallizes in the body during disease, obtaining high-quality crystals suitable for structural analysis previously required recrystallization outside the body.
Next, the Gal-10 crystals obtained were soaked in a sugar molecule solution, allowing sugar molecules to be incorporated into the crystals, and X-ray crystal structure analysis was performed.
As a result, the researchers were able to reveal the structures of disaccharides and trisaccharides. These are small molecules consisting of monosaccharides linked by glycosidic bonds. As the rotation of these bonds causes the molecular structure to constantly fluctuate, it was considered difficult to orient them uniformly for their structural analysis. Notably, the group determined the three-dimensional structure of the trisaccharide melezitose at atomic resolution for the first time in the world.
Furthermore, they created a mutant (E33A mutant) with slight modifications to the sugar-binding site of Gal-10, performed X-ray crystal structure analysis of melezitose, and conducted fluctuation analysis of the resulting the crystal structure data. The results showed that melezitose exhibited large fluctuations within wild-type Gal-10, while it was more immobilized in the E33A mutant. Additionally, they found that in the wild type, the fluctuations of surrounding amino acids increased upon melezitose binding, whereas in the E33A mutant, they decreased.
Molecular dynamics simulations using the supercomputer "TSUBAME4.0" showed that in the wild type, melezitose had more possible structural variations than in the E33A mutant, transitioning between multiple structures. This revealed that control of the "fluctuations" of sugar molecules depends on the fine structure of proteins.
In the future, by extending the application of this technology beyond sugar molecules, the group plans to deepen the systematic understanding of protein-small molecule interactions.
Ueno commented: "Simultaneously capturing the 'structure' and 'fluctuations' of molecules that move flexibly like sugars has been a long-standing challenge. This technology serves as a new platform to achieve this. Because we can understand at the atomic level how various molecules that serve as the seeds for medicines bind and move in the body, I am confident that this will greatly accelerate efficient drug design and contribute to drug discovery research."
Journal Information
Publication: Small Structures
Title: Cell-Free Protein Crystallization Enables Rapid Structure Determination of Disaccharides and Trisaccharides Using Galectin-10 Crystals
DOI: 10.1002/sstr.202500501
This article has been translated by JST with permission from The Science News Ltd. (https://sci-news.co.jp/). Unauthorized reproduction of the article and photographs is prohibited.