Involvement of Histidine Residues in the pH-Dependent β-Galactoside Binding Activity of Human Galectin-1
Involvement of Histidine Residues in the pH-Dependent β-Galactoside Binding Activity of Human Galectin-1
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组氨酸残基参与 pH 依赖性 β-半乳糖苷结合活性的人 Galectin-1
DOI：
10.1021/bi4001112
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发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
and Hideo Takeuchi
中科院分区：
文献类型：
--
作者：
Hirotsugu Hiramatsu;Katsuyuki Takeuchi;and Hideo Takeuchi
以乳糖为配体，用荧光光谱法研究了人半乳糖凝集素-1(hGal-1)与β-半乳糖苷结合活性的pH依赖性。在pH为7.5时，结合常数Kb值为2.94±0.10 mm-1。Kb值在酸性pH下下降，在pH 6.0±0.1处有一个转变中点。为了阐明pH依赖性的分子机制，我们用荧光、圆二色谱、紫外吸收和紫外共振拉曼光谱研究了hGal-1及其两个组氨酸突变体(H44Q和H52Q)的结构。光谱分析表明，His44和His52的pKavue分别为5.7±0.2和6.3±0.1。His52在pH 6.3以下的质子化引起二级结构的微小变化，并部分降低了半乳糖苷的结合活性。另一方面，His44在pH 5.7以下的质子化作用与Trp68发生阳离子−π相互作用，大大降低了半乳糖苷的结合活性。参考文献在pH7.0和5.6时的X射线结构，质子化的His52被认为是随着含有His52的β链的部分展开而略远离半乳糖苷结合区。另一方面，质子化的His44不能与半乳糖苷形成氢键，并通过阳离子−π相互作用诱导Trp68的重取向和/或位移，导致半乳糖苷结合口袋的松动。这些与His质子化相关的结构变化很可能是hGal-1的半乳糖苷结合活性的pH依赖性的起源。
The pH dependence of the β-galactoside binding activity of human galectin-1 (hGal-1) was investigated by fluorescence spectroscopy using lactose as a ligand. The obtained binding constantKbwas 2.94 ± 0.10 mM–1at pH 7.5. TheKbvalue decreased at acidic pH with a midpoint of transition at pH 6.0 ± 0.1. To elucidate the molecular mechanism of the pH dependence, we investigated the structures of hGal-1 and its two His mutants (H44Q and H52Q) using fluorescence, circular dichroism, UV absorption, and UV resonance Raman spectroscopy. Analysis of the spectra has shown that the pKavalues of His44 and His52 are 5.7 ± 0.2 and 6.3 ± 0.1, respectively. The protonation of His52 below pH 6.3 induces a small change in secondary structure and partly reduces the galactoside binding activity. On the other hand, the protonation of His44 below pH 5.7 exerts a cation−π interaction with Trp68 and largely diminishes the galactoside binding activity. With reference to the literature X-ray structures at pH 7.0 and 5.6, protonated His52 is proposed to move slightly away from the galactoside-binding region with a partial unfolding of the β-strand containing His52. On the other hand, protonated His44 becomes unable to form a hydrogen bond with galactoside and additionally induces a reorientation and/or displacement of Trp68 through cation−π interaction, leading to a loosening of the galactoside-binding pocket. These structural changes associated with His protonation are likely to be the origin of the pH dependence of the galactoside binding activity of hGal-1.