Kozai Daisuke, Inoue Masao, Suzuki Shota, Kamegawa Akiko, Nishikawa Kouki, Suzuki Hiroshi, Ekimoto Toru, Ikeguchi Mitsunori, Fujiyoshi Yoshinori
Cellular and Structural Physiology Laboratory (CeSPL), Advanced Research Initiative, Institute of Integrated Research, Institute of Science Tokyo, Tokyo, Japan.
Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.
Nat Commun. 2025 Mar 20;16(1):2653. doi: 10.1038/s41467-025-57728-3.
Aquaglyceroporins such as aquaporin-3 (AQP3) and its bacterial homologue GlpF facilitate water and glycerol permeation across lipid bilayers. X-ray crystal structures of GlpF showed open pore conformations, and AQP3 has also been predicted to adopt this conformation. Here we present cryo-electron microscopy structures of rat AQP3 and GlpF in different narrowed pore conformations. In n-dodecyl-β-D-maltopyranoside detergent micelles, aromatic/arginine constriction filter residues of AQP3 containing Tyr212 form a 2.8-Å diameter pore, whereas in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) nanodiscs, Tyr212 inserts into the pore. Molecular dynamics simulation shows the Tyr212-in conformation is stable and largely suppresses water permeability. AQP3 reconstituted in POPC liposomes exhibits water and glycerol permeability, suggesting that the Tyr212-in conformation may be altered during permeation. AQP3 Y212F and Y212T mutant structures suggest that the aromatic residue drives the pore-inserted conformation. The aromatic residue is conserved in AQP7 and GlpF, but neither structure exhibits the AQP3-like conformation in POPC nanodiscs. Unexpectedly, the GlpF pore is covered by an intracellular loop, but the loop is flexible and not primarily related to the GlpF permeability. Our findings illuminate the unique AQP3 conformation and structural diversity of aquaglyceroporins.
水甘油通道蛋白，如 aquaporin-3（AQP3）及其细菌同源物 GlpF，可促进水和甘油透过脂质双层。GlpF 的 X 射线晶体结构显示为开放孔构象，AQP3 也被预测为采用这种构象。在此，我们展示了大鼠 AQP3 和 GlpF 在不同狭窄孔构象下的冷冻电子显微镜结构。在正十二烷基-β-D-麦芽糖苷去污剂胶束中，含有 Tyr212 的 AQP3 的芳香族/精氨酸收缩过滤残基形成了直径为 2.8 埃的孔，而在 1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）纳米盘中，Tyr212 插入孔中。分子动力学模拟表明 Tyr212 插入构象是稳定的，并在很大程度上抑制了水的渗透性。重构在 POPC 脂质体中的 AQP3 表现出水和甘油的渗透性，这表明 Tyr212 插入构象在渗透过程中可能会发生改变。AQP3 Y212F 和 Y212T 突变体结构表明芳香族残基驱动孔插入构象。芳香族残基在 AQP7 和 GlpF 中是保守的，但在 POPC 纳米盘中，这两种结构均未表现出 AQP3 样构象。出乎意料的是，GlpF 孔被一个细胞内环覆盖，但该环是灵活的，且与 GlpF 的渗透性无主要关联。我们的研究结果揭示了 AQP3 的独特构象以及水甘油通道蛋白的结构多样性。
