Nuclear Magnetic Resonance Structural Characterization of Substrates Bound to the α-2,6-Sialyltransferase, ST6Gal-I
Nuclear Magnetic Resonance Structural Characterization of Substrates Bound to the α-2,6-Sialyltransferase, ST6Gal-I
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DOI：
10.1021/bi9015154
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发表时间：
2009-12-01
期刊：
影响因子：
2.9
通讯作者：
Prestegard, James H.
中科院分区：
文献类型：
--
作者：
Liu, Shan;Meng, Lu;Prestegard, James H.
α-2，6-唾液酸转移酶（ST 6 Gal-I）是调节哺乳动物细胞表面含唾液酸分子分布的关键酶。然而，其天然形式是膜结合和糖基化的事实使得通过X射线晶体学对这种真核蛋白进行结构表征变得困难。它的大尺寸（仅催化结构域就接近40 kDa）也对通过核磁共振（NMR）进行完整结构测定提出了挑战。然而，即使没有完整的结构确定，也有NMR策略可以返回有关蛋白质选择区域的目标信息，包括从其结合配体的角度来看的活性位点的信息。在这里，在以前的研究的延续，自旋标记的模拟的聚糖受体配体被用来确定位于蛋白质活性位点的其他氨基酸。此外，自旋标记的供体用于表征两个结合配体的相对位置。用转移核Overhauser效应（trNOE）和饱和转移差（STD）实验研究了配体构象和蛋白质-配体接触表面。由上述方法提供的数据导致结合底物的几何模型，其在许多方面携带ST 6 Gal-I结合位点的印记。
The alpha-2,6-sialyltransferase (ST6Gal-I) a key enzyme that regulates the distribution of sialic acid-containing molecules on mammalian cell surfaces. However, the fact that its native form is membrane-bound and glycosylated has made structural characterization by X-ray crystallography of this eukaryotic protein difficult. Its large size (similar to 40 kDa for just the catalytic domain) also poses a challenge for complete structure determination by nuclear magnetic resonance (NMR). However, even without complete structure determination, there are NMR strategies that can return targeted information about select regions of the protein, including information about the active site as seen from the perspective of its bound ligands. Here, in a continuation of a previous study, a spin-labeled mimic of a glycan acceptor ligand is used to identify additional amino acids located in the protein active site. In addition, the spin-labeled donor is used to characterize the relative placement of the two bound ligands. The ligand conformation and protein-ligand contact Surfaces are studied by transferred nuclear Overhauser effects (trNOEs) and saturation transfer difference (STD) experiments. The data afforded by the methods mentioned above lead to a geometric model of the bound substrates that in many ways carries an imprint of the ST6Gal-I binding site.