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首页> 外文期刊>Biochemistry >The ice-binding site of sea raven antifreeze protein is distinct from the carbohydrate-binding site of the homologous C-type lectin
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The ice-binding site of sea raven antifreeze protein is distinct from the carbohydrate-binding site of the homologous C-type lectin

机译：海鸦防冻蛋白的冰结合位点不同于同源C型凝集素的碳水化合物结合位点

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摘要

Antifreeze proteins lower the freezing point of their solution by binding to ice and inhibiting its growth. One of several structurally different antifreeze proteins in fishes (type II) is homologous to the carbohydrate-recognition domain of Ca2+-dependent lectins and adopts the same three-dimensional fold. Type II antifreeze proteins from herring and smelt require Ca2+ for binding to ice, whereas this same antifreeze protein in sea raven binds to ice in the absence of Ca2+ and has only two of the five Ca2+-liganding amino acids that are present in the lectin. To locate the ice-binding site, site-directed mutants of the 15 kDa, globular, disulfide-bonded sea raven antifreeze protein were produced by secretion from Pichia pastoris. Pairs of amino acid replacements, insertions, and a peptide loop swap were made in the region equivalent to the sugar-binding site of the lectin that encompasses loops 3 and 4 and beta-sheets 7 and 8. Even the most extensive mutation caused only a 25% decrease in antifreeze activity and demonstrated that the residues corresponding to the Ca2+-binding site are only peripherally involved in ice binding, When adjacent surface residues were mutated, the replacement of one residue, Ser120 by His, caused a 35% decrease in activity by itself and an 80% loss in conjunction with the peptide loop swap mutation, This pivotal sea raven antifreeze protein amino acid does not coincide with the herring ice-binding epicenter, but is located within the region corresponding to the proposed CaCO3-binding surface of a third homologue, the pancreatic stone protein. Intron and exon structure of the sea raven AFP gene also suggests that it might be more closely related to the stone protein gene than to the lectin gene. These results support the notion that this family of proteins has evolved more than one binding surface from the same protein scaffold. [References: 54]
机译：抗冻蛋白通过与冰结合并抑制其生长来降低其溶液的凝固点。鱼类（II型）中几种结构不同的抗冻蛋白之一与Ca2 +依赖的凝集素的碳水化合物识别域同源，并具有相同的三维折叠。来自鲱鱼和熔炼的II型抗冻蛋白需要Ca2 +才能与冰结合，而在海乌鸦中，这种相同的抗冻蛋白在没有Ca2 +的情况下与冰结合，并且凝集素中只有五个结合Ca2 +的氨基酸中的两个。为了定位冰结合位点，通过巴斯德毕赤酵母的分泌产生了15 kDa，球形，二硫键结合的海鸦抗冻蛋白的定点突变体。在相当于凝集素的糖结合位点的区域中进行了成对的氨基酸置换，插入和肽环交换，该区域包括环3和4以及β-sheets7和8。即使最广泛的突变也仅导致了抗冻活性降低25％，并且证明与Ca2 +结合位点相对应的残基仅在外围参与冰的结合。当相邻的表面残基发生突变时，用His替换一个残基Ser120会使活性降低35％它本身以及与肽环交换突变相关的80％的损失，这种关键的海鸦抗冻蛋白氨基酸与鲱鱼的冰结合震中并不重合，而是位于与拟议的CaCO3结合表面相对应的区域内第三个同源物，胰石蛋白。乌鸦AFP基因的内含子和外显子结构也表明，它可能与石蛋白基因比与凝集素基因更紧密相关。这些结果支持了这一蛋白质家族已经从相同的蛋白质支架进化出一个以上结合表面的观点。 [参考：54]

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