Ca2+-dependent antifreeze proteins - Modulation of conformation and activity by divalent metal ions
Ca2+-dependent antifreeze proteins - Modulation of conformation and activity by divalent metal ions
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DOI：
10.1074/jbc.271.28.16627
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发表时间：
1996-07-12
影响因子：
4.8
通讯作者：
Hew, CL
中科院分区：
文献类型：
--
作者：
Ewart, KV;Yang, DSC;Hew, CL
抗冻蛋白（AFP）是一种结构多样的分子，它们具有与冰晶结合并抑制冰晶生长的能力。大西洋鲱鱼和胡瓜鱼的II型鱼AFP在已知的AFP中是独特的，因为它们需要一种辅助因子来进行抗冻活性。这些AFP与Ca 2+依赖性（C型）凝集素的碳水化合物识别结构域同源，并且需要Ca 2+来实现其活性。为了研究金属离子在II型AFP结构和功能中的作用，研究了Ca 2+和其他二价阳离子与鲱鱼AFP的结合。用Ca-45（2+）结合研究表明，AFP有一个单一的Ca ~（2+）结合位点，Kd为9 μ M。蛋白水解保护的研究和抗冻活性的测量揭示了从蛋白酶敏感的和非活性的apoAFP的蛋白酶抗性活性AFP后的Ca 2+结合的构象变化。其他二价金属离子包括Mn 2+、Ba 2+和Zn 2+结合在Ca 2+结合位点并诱导类似的变化。在340 nm处色氨酸发射强度的饱和增加也发生在Ca ~（2+）添加后，而抗冻活性似乎正常时，Ca ~（2+）或Mn ~（2+）的约束，它是在其他金属离子的存在下低得多。当Ba ~（2+）与AFP结合时，冰晶的形态表现出明显的差异。这些研究表明，鲱鱼甲胎蛋白特异性结合钙离子，因此，采用的构象是必不可少的冰结合活性。
The antifreeze proteins (AFPs) are structurally diverse molecules that share an ability to bind to ice crystals and inhibit their growth, The type II fish AFPs of Atlantic herring and smelt are unique among known AFPs in their requirement of a cofactor for antifreeze activity. These AFPs are homologous with the carbohydrate-recognition domains of Ca2+-dependent (C-type) lectins and require Ca2+ for their activity. To investigate the role of metal ions in the structure and function of type II AFPs, the binding of Ca2+ and other divalent cations to herring AFP was investigated. Binding studies using Ca-45(2+) demonstrated that the AFP has a single Ca2+-binding site with a K-d of 9 mu M. Proteolysis protection studies and measurement of antifreeze activity revealed a conformational change from a protease-sensitive and inactive apoAFP to a protease resistant active AFP upon Ca2+ binding. Other divalent metal ions including Mn2+, Ba2+, and Zn2+ bind at the Ca2+-binding site and induce a similar change. A saturatable increase in tryptophan emission intensity at 340 nm also occurred upon Ca2+ addition, Whereas antifreeze activity appeared normal when Ca2+ or Mn2+ were bound, it was much lower in the presence of other metal ions. When Ba2+ was bound to the AFP, ice crystals showed a distinct difference in morphology. These studies demonstrate that herring AFP specifically binds Ca2+ and, consequently, adopts a conformation that is essential for its ice binding activity.