The structure of the catechin-binding site of human sulfotransferase 1A1
The structure of the catechin-binding site of human sulfotransferase 1A1
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DOI：
10.1073/pnas.1613913113
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发表时间：
2016-12-13
影响因子：
11.1
通讯作者：
Leyh, Thomas S.
中科院分区：
文献类型：
--
作者：
Cook, Ian;Wang, Ting;Leyh, Thomas S.
我们刚刚开始了解人类细胞质硫转移酶（SULTs）家族13疾病相关酶的变构调节，这些酶调节数百种（如果不是数千种的话）信号小分子的活动。SULT1A1是成人肝脏中的主要亚型，包含两个不相互作用的变构位点，每个位点结合不同的分子家族：儿茶素（天然存在的黄酮醇）和非甾体抗炎药（NSAIDs）。在这里，我们展示了SULT变构结合位点的结构- SULT1A1与表没食子儿茶素没食子酸酯（EGCG）结合的儿茶素结合位点。变构口袋位于蛋白质的动态区域，使EGCG能够控制酶活性位点帽的打开和关闭。此外，该结构为EGCG的同工酶特异性提供了分子解释，实验证实了这一点。结合位点结构是在没有x射线晶体学或多维核磁共振的情况下获得的。相反，使用SULT1A1载子蛋白结构来指导少量自旋标记的单cysmutants的定位，这些自旋标记的单cysmutants用足够强度的顺磁场覆盖整个酶表面，以确定其对结合配体的横向（T-2）弛弛性的贡献。利用T-2值进行三角测量，将EGCG质子映射到蛋白质表面，以计算它们与三个自旋标记的Cys突变体的距离。通过距离约束分子动力学对接得到最终结构。这种方法很容易扩展到其他系统，适用于大范围的配体亲和，需要很少的蛋白质，避免了对同位素标记蛋白质的需要，并且没有蛋白质分子量的限制。
We are just beginning to understand the allosteric regulation of the human cytosolic sulfotransferase (SULTs) family-13 disease-relevant enzymes that regulate the activities of hundreds, if not thousands, of signaling small molecules. SULT1A1, the predominant isoform in adult liver, harbors two noninteracting allosteric sites, each of which binds a different molecular family: the catechins (naturally occurring flavonols) and nonsteroidal antiinflammatory drugs (NSAIDs). Here, we present the structure of an SULT allosteric binding site-the catechin-binding site of SULT1A1 bound to epigallocatechin gallate (EGCG). The allosteric pocket resides in a dynamic region of the protein that enables EGCG to control opening and closure of the enzyme's active-site cap. Furthermore, the structure offers a molecular explanation for the isozyme specificity of EGCG, which is corroborated experimentally. The binding-site structure was obtained without X-ray crystallography or multidimensional NMR. Instead, a SULT1A1 apoprotein structure was used to guide positioning of a small number of spin-labeled single-Cysmutants that coat the entire enzyme surface with a paramagnetic field of sufficient strength to determine its contribution to the bound ligand's transverse (T-2) relaxation from its 1D solution spectrum. EGCG protons were mapped to the protein surface by triangulation using the T-2 values to calculate their distances to a trio of spin-labeled Cys mutants. The final structure was obtained using distance-constrained molecular dynamics docking. This approach, which is readily extensible to other systems, is applicable over a wide range of ligand affinities, requires little protein, avoids the need for isotopically labeled protein, and has no protein molecular weight limitations.