Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen-Deuterium Exchange
Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen-Deuterium Exchange
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DOI：
10.3390/molecules22081357
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发表时间：
2017-08-01
期刊：
影响因子：
4.6
通讯作者：
Hsu, Shang-Te Danny
中科院分区：
文献类型：
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作者：
Chien, Chih-Ta Henry;Ho, Meng-Ru;Hsu, Shang-Te Danny
半乳糖凝集素是一种与β -半乳糖苷结合的蛋白，与多种生物功能有关。尽管它们的碳水化合物结合基序高度保守，结构基本相同，但它们对乳糖（一种常见的凝集素抑制剂）的亲和力差异很大。在这里，我们的目的是使用基于溶液的技术来研究乳糖凝集素之间的乳糖亲和差异的分子基础。通过核磁共振（NMR）光谱、本征色氨酸荧光、等温滴定量热法和生物层干涉法对人半乳糖凝集素-1 （hGal1）、半乳糖凝集素-7 （hGal7）和半乳糖凝集素-8的n端和c端结构域（分别为hGal8NTD和hGal8CTD）得到了乳糖结合的一致解离常数。此外，从核磁共振线形分析中提取了乳糖结合的解离率。化学位移扰动的结构映射揭示了hGal1和hGal8NTD在乳糖结合上的远程扰动。我们进一步利用核磁共振的氢-氘交换（HDX）证明，乳糖结合增加了hGal1和hGal8NTD的配体结合袋的另一侧残基的交换速率，表明了变构。此外，乳糖结合诱导了hGal8CTD在整个结构域的显著稳定性。我们的研究结果表明，乳糖结合在非常慢的时间尺度上（几分钟或更慢）降低了hGal8CTD的内部动力学，代价是由于不利的构象熵损失而降低了结合亲和力。
Galectins are beta-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their affinities for lactose, a common galectin inhibitor, vary significantly. Here, we aimed to examine the molecular basis of differential lactose affinities amongst galectins using solution-based techniques. Consistent dissociation constants of lactose binding were derived from nuclear magnetic resonance (NMR) spectroscopy, intrinsic tryptophan fluorescence, isothermal titration calorimetry and bio-layer interferometry for human galectin-1 (hGal1), galectin-7 (hGal7), and the N-terminal and C-terminal domains of galectin-8 (hGal8NTD and hGal8CTD, respectively). Furthermore, the dissociation rates of lactose binding were extracted from NMR lineshape analyses. Structural mapping of chemical shift perturbations revealed long-range perturbations upon lactose binding for hGal1 and hGal8NTD. We further demonstrated using the NMR-based hydrogen-deuterium exchange (HDX) that lactose binding increases the exchange rates of residues located on the opposite side of the ligand-binding pocket for hGal1 and hGal8NTD, indicative of allostery. Additionally, lactose binding induces significant stabilisation of hGal8CTD across the entire domain. Our results suggested that lactose binding reduced the internal dynamics of hGal8CTD on a very slow timescale (minutes and slower) at the expense of reduced binding affinity due to the unfavourable loss of conformational entropy.