—Revealing the key role of the hydrophobic pocket—

A joint research group consisting of Hikaru Ichida, a doctoral student in the Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University; Kosuke Mizuno, currently a Postdoctoral Researcher at the Institute for Protein Research, The University of Osaka; Professor Noriyuki Kodera and Associate Professor Holger Flechsig of the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University; and Associate Professor Satoshi Toda of the Institute for Protein Research, The University of Osaka, has succeeded in visualizing the structural dynamics underlying how the serum protein Afamin stabilizes and transports Wnt3a, a lipid-modified signaling molecule. The study also showed that stable binding between these two molecules depends on both a hydrophobic pocket that accommodates Wnt3a and the structural integrity of Afamin.
Wnt proteins are essential molecules that help the body develop properly and maintain healthy tissues. However, because they do not dissolve well in water and are highly hydrophobic, they tend to be unstable in the body. This study has revealed part of the mechanism by which Wnt3a is stably transported with the help of another protein. These findings are expected to deepen our understanding of biological processes involving Wnt3a and may contribute in the future to the development of ex vivo tissue engineering technologies and regenerative medicine.
The results of this study was published in the online edition of Nano Letters on April 15th, 2026.

【Background】
Wnt proteins are signaling molecules that play important roles in morphogenesis and stem cell maintenance. Because Wnt proteins are modified with lipids, they are highly hydrophobic and cannot remain stable on their own in water. For this reason, understanding how they are stabilized and transported in aqueous environments has been an important question.
Afamin is a glycoprotein found in blood serum and is known as a carrier protein for fat-soluble molecules. Previous studies showed that Afamin forms a complex with Wnt3a and keeps it soluble while preserving its biological activity. However, it was still unclear how these two molecules are arranged in three dimensions and how they move together. In particular, the role of the hydrophobic pocket (*1) in the central part of Afamin in Wnt3a transport had not been clarified.
【Summary of the Research Findings】
The research group first used high-speed AFM (*2) to observe single molecules and found that Afamin shows a hinge-like opening and closing motion between its two globular domains, one large and one small (Fig. 1). In addition, molecular modeling showed that this flexible motion comes from the structure in the central part of Afamin (Fig. 2).
Next, the group observed the Afamin–Wnt3a complex (Afamin/Wnt3a) by high-speed AFM and, for the first time in the world, successfully captured the appearance of a single complex molecule that had previously been difficult to observe directly. They found that the complex does not have just one fixed conformation. Instead, it takes two forms: a symmetric structure, in which Wnt3a is located near the center, and an asymmetric structure, in which Wnt3a is shifted to one side. These two forms were seen to change into each other. The researchers also found that when Wnt3a binds to Afamin, the overall fluctuation of the molecule becomes smaller than that of Afamin alone, showing that its structural flexibility is reduced (Fig. 3).
Furthermore, cell-based experiments revealed that afamin mutants—with substitutions in amino acids associated with the hydrophobic pocket—failed to interact with Wnt3a on the cell surface. These results suggest that the structural integrity of the hydrophobic pocket is essential for the formation of the afamin-Wnt3a complex. (Fig. 4).
In this study, the researchers used a unique microscopy technology, one of the strengths of WPI-NanoLSI at Kanazawa University, as the core method to directly observe molecular motion and capture shape changes in protein complexes moving in solution. In addition, integrative modelling was used to infer three-dimensional model structures of the proteins from HS-AFM images, helping the researchers to provide atomistic understanding of measurement data. By further combining this with cell-based binding experiments, the study provided new structural insight into how lipid-modified proteins are transported.

【Future Perspectives】
This study suggests that Afamin may function not simply as a holder of Wnt3a, but as a carrier that transports it while dynamically changing its structure. In the future, direct observation of how Wnt3a is passed from Afamin to Wnt receptors or other binding partners is expected to help clarify the molecular mechanism of Wnt transport outside the cell.

This work was supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan. Also, this work was supported by KAKENHI (24H00402), JPSP, Japan, PRESTO (JPMJPR23J2), JST, Japan, WISE Program of Kanazawa University by MEXT, JST, the establishment of university fellowships towards the creation of science technology innovation (JPMJFS2116), and JST SPRING (JPMJSP2135), Japan, Yoshida Scholarship Foundation and The ANRI Fellowship.

【Article】
Journal：Nano Letters
Title：Structural Dynamics of the Afamin/Wnt3a Complex Mediated by the Afamin Hydrophobic Pocket
Author：Hikaru Ichida, Kosuke Mizuno, Romain Amyot, Kenichi Umeda, Satoshi Toda, Holger Flechsig, Noriyuki Kodera
Publication date：Apr 15, 2026
DOI：10.1021/acs.nanolett.5c05561
URL：https://doi.org/10.1021/acs.nanolett.5c05561

【Glossary】
*1　Hydrophobic pocket
A structural region that can accommodate molecules that do not dissolve well in water. In this study, this pocket was found inside Afamin and shown to play an important role in holding the lipid part of Wnt3a.

*2　High-speed AFM
A unique microscopy technique that can capture the shapes and movements of biomolecules such as proteins in liquid as video. It can record molecular motion in real time with a time resolution of about 0.1 seconds (10 frames per second). Its spatial resolution is about 1 nm in the horizontal (XY) direction and about 0.1 nm in the vertical (Z) direction, which is high enough to distinguish protein domain structures.

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【Contact】
■Research Inquiries
Noriyuki Kodera, Professor
Nano Life Science Institute (WPI-NanoLSI), Kanazawa University
E-mail: nkodera@staff.kanazawa-u.ac.jp

Holger Flechsig, Associate Professor
Nano Life Science Institute (WPI-NanoLSI), Kanazawa University
E-mail: flechsig@staff.kanazawa-u.ac.jp

Satoshi Toda, Associate Professor
Institute for Protein Research, The University of Osaka
E-mail: satoshi.toda@protein.osaka-u.ac.jp

■Media inquiries
Masuo Goto, Associate professor
Nano Life Science Institute (WPI-NanoLSI), Kanazawa University
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