STRUCTURE-FUNCTION RELATIONSHIP IN THE GLOBULAR TYPE-III ANTIFREEZE PROTEIN - IDENTIFICATION OF A CLUSTER OF SURFACE RESIDUES REQUIRED FOR BINDING TO ICE
STRUCTURE-FUNCTION RELATIONSHIP IN THE GLOBULAR TYPE-III ANTIFREEZE PROTEIN - IDENTIFICATION OF A CLUSTER OF SURFACE RESIDUES REQUIRED FOR BINDING TO ICE
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DOI：
10.1002/pro.5560031016
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发表时间：
1994-10-01
期刊：
影响因子：
8
通讯作者：
DAVIES, PL
中科院分区：
文献类型：
--
作者：
CHAO, H;SONNICHSEN, FD;DAVIES, PL
抗冻蛋白（AFPs）通过结合和抑制冰的生长来降低水溶液的冰点。一些鱼类AFP的冰结表面是由它们的线性、重复的氢键基序表明的，而66个氨基酸长的III型AFP具有紧凑的球状褶皱，没有任何明显的周期性。在结构中，9条β -链配对形成2条三链反平行片和1条双链反平行片，2条三链反平行片排列成正交β -三明治（Sonnichsen FD, Sykes BD, Chao H, Davies FL, 1993, Science:1154-1157）。根据其结构和III型AFP异构体序列的排列，在靠近c端的三链片上及其周围发现了一簇保守的、极性的、表面可接近的氨基酸（N14、T18、Q44和N46）。在其中3个位点上，改变酰胺和羟基的突变导致抗冻活性大幅下降，但酰胺到羧酸的变化产生了在pH 3和pH 6下完全活跃的AFPs。这与观察结果一致，即III型AFP在pH 2至pH 11时具有最佳活性。在浓度为1 mg/mL时，Q44T、N14S和T18N的活性分别为野生型抗冻液的50%、25%和10%。突变的影响是累积的，双突变体N14S/Q44T具有10%的野生型活性，而三突变体N14S/T18N/Q44T没有活性。所有活性降低的突变体都被核磁共振证明是正确折叠的。此外，通过二维核磁共振光谱对三重突变体的完整表征表明，单个突变和组合突变并没有显著改变这些蛋白质的结构。这些结果表明III型AFP的c端β -sheet主要负责抗冻活性，并且他们确定N14， T18和444是AFP-ice相互作用的关键残基。
Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice-binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66-amino-acid-long Type III AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 beta-strands are paired to form 2 triple-stranded antiparallel sheets and 1 double-stranded antiparallel sheet, with the 2 triple sheets arranged as an orthogonal beta-sandwich (Sonnichsen FD, Sykes BD, Chao H, Davies FL, 1993, Science 259:1154-1157). Based on its structure and an alignment of Type III AFP isoform sequences, a cluster of conserved, polar, surface-accessible amino acids (N14, T18, Q44, and N46) was noted on and around the triple-stranded sheet near the C-terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type III AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50%, 25%, and 10% of the activity of wild-type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild-type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2-dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C-terminal beta-sheet of Type III AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and 444 as key residues for the AFP-ice interaction.