Graduate School of Life Science, Hokkaido University, Sapporo, Japan; Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Sapporo, Japan.
FEBS J. 2014 Aug;281(16):3576-90. doi: 10.1111/febs.12878. Epub 2014 Jul 4.
Antifreeze proteins (AFPs) are structurally diverse macromolecules that bind to ice crystals and inhibit their growth to protect the organism from injuries caused by freezing. An AFP identified from the Antarctic bacterium Colwellia sp. strain SLW05 (ColAFP) is homologous to AFPs from a wide variety of psychrophilic microorganisms. To understand the antifreeze function of ColAFP, we have characterized its antifreeze activity and determined the crystal structure of this protein. The recombinant ColAFP exhibited thermal hysteresis activity of approximately 4 °C at a concentration of 0.14 mm, and induced rapid growth of ice crystals in the hexagonal direction. Fluorescence-based ice plane affinity analysis showed that ColAFP binds to multiple planes of ice, including the basal plane. These observations show that ColAFP is a hyperactive AFP. The crystal structure of ColAFP determined at 1.6 Å resolution revealed an irregular β-helical structure, similar to known homologs. Mutational and molecular docking studies showed that ColAFP binds to ice through a compound ice-binding site (IBS) located at a flat surface of the β-helix and the adjoining loop region. The IBS of ColAFP lacks the repetitive sequences that are characteristic of hyperactive AFPs. These results suggest that ColAFP exerts antifreeze activity through a compound IBS that differs from the characteristic IBSs shared by other hyperactive AFPs. This study demonstrates a novel method for protection from freezing by AFPs in psychrophilic microorganisms.
Structural data for ColAFP have been submitted to the Protein Data Bank (PDB) under accession number 3WP9.
抗冻蛋白（AFPs）是结构多样的大分子，它们与冰晶结合并抑制其生长，从而保护生物体免受冻结造成的伤害。从南极细菌 Colwellia sp. 菌株 SLW05（ColAFP）中鉴定出的 AFP 与来自各种嗜冷微生物的 AFP 同源。为了了解 ColAFP 的抗冻功能，我们对其抗冻活性进行了表征，并确定了该蛋白的晶体结构。重组 ColAFP 在 0.14mm 的浓度下表现出约 4°C 的热滞活性，并诱导冰晶在六方方向上快速生长。基于荧光的冰面亲和力分析表明，ColAFP 结合到多个冰面，包括基面。这些观察结果表明，ColAFP 是一种超活性 AFP。在 1.6Å 分辨率下确定的 ColAFP 晶体结构显示出不规则的β-螺旋结构，与已知同源物相似。突变和分子对接研究表明，ColAFP 通过位于β-螺旋和平坦表面的复合冰结合位点（IBS）与冰结合。ColAFP 的 IBS 缺乏超活性 AFP 所具有的重复序列。这些结果表明，ColAFP 通过与其他超活性 AFP 共享的特征 IBS 不同的复合 IBS 发挥抗冻活性。本研究证明了嗜冷微生物中 AFP 防止冻结的一种新方法。
ColAFP 的结构数据已提交给蛋白质数据库（PDB），登录号为 3WP9。
