"人類半乳糖凝集素1, 7, 8之乳糖結合親和力結構分析"

Abstract

半乳糖凝集素是一個特定辨識半乳糖衍生物的凝集素家族。它們表現在細胞質、細胞核、以及胞外，並且擁有各式不同的生物功能。研究顯示半乳糖凝集素跟細胞間作用、細胞基質吸附、跨膜信號息息相關。目前已經發現十五種半乳糖凝集素，它們的序列相似度並不高，對於乳糖的結合能力也非常不同，但三維結構，無論是骨架或支鏈結構幾乎一模一樣。即使是直接接觸配體的支鏈結構也都難以用肉眼分辨，所以基質辨識的機制很難從晶體結構解釋。我們使用三種不需要額外螢光標記的技術（自身螢光、干涉儀、核磁共振）來量測半乳糖凝集素與乳糖的結合強度，如此可以避免螢光標記所造成的誤差， 增加數值的可信度。為了要解釋半乳糖凝集素是如何辨識基質以及它的選擇性，我們系統性的研究半乳糖凝集素一號、七號、及八號在結合乳糖後對於動態及折疊穩定性的影響。我們使用的工具包括分子動力模擬、圓二色光譜儀、螢光光譜儀、以及核磁共振光譜儀。我們使用核磁共振光譜儀來監測氫氘交換的速率，藉由觀察乳糖結合後交換速率的改變，我們可以獲得原子級解析度的結構及穩定性的資訊。與乳糖結合不僅影響直接接觸的殘基，一些與結合位置較遠的氨基酸也有明顯的改變。螢光及圓二色光譜加上奇異值分解的輔助來偵測平衡狀態下半乳糖凝集素的摺疊穩定性。半乳糖凝集素一號、七號、八號氮端區段、及八號碳端區段有非常不同的折疊特性：一號及八號氮端區段是兩態摺疊機制而七號及八號碳端是三態摺疊機制。這樣不同的特性或許可以解釋它們對於基質有不同的結合能力及選擇性。我們的實驗結果對於設計高專一性及高結合性的藥物將會有很大的幫助。

Galectins are a family of lectins that interact with β-galactosides, express in cytosol, nucleus, and extracellular matrix, and confer a broad range of functions including cell-cell interaction, cell-matrix adhesion and transmembrane signaling. Despite their low sequence homology, their three-dimensional structures are essentially identical. The binding affinity of galectins to lactose and other β-galactosides vary significantly. However, it is difficult to rationalize the molecular recognition mechanisms of different galectins based on their crystal structures of the apo- and holo-forms, which exhibit minimal conformational changes even for the side-chains that are involved in ligand binding. In order to better understand the origin of substrate binding affinity and selectivity, we systematically investigate the differential effects of lactose binding on human galectin-1, -7, and -8 in terms of their internal dynamics and folding stabilities using molecular dynamics (MD) simulation, far-UV circular dichroism (CD), intrinsic fluorescence and nuclear magnetic resonance (NMR) spectroscopy. NMR hydrogen-deuterium exchange (HDX) data suggest that lactose binding not only perturbs the structures and dynamics of the residues that are directly involved ligand binding but also results in long-range perturbations at the dimer interface for galectin-1 and -7. This is consistent with the differences in fast internal dynamics in MD simulations and 15N spin relaxation dynamics, as well as the folding stabilities of galectin-1, -7, -8 in response to lactose binding. Such differential responses to ligand binding for different galectins may be implicated in modulating the binding affinity and selectivity, and hence could potentially be exploited for designs of better inhibitors with higher specificity.