Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461-1926, United States.
Biochemistry. 2022 May 17;61(10):843-855. doi: 10.1021/acs.biochem.1c00740. Epub 2022 May 6.
Among human cytosolic sulfotransferases, SULT2B1b is highly specific for oxysterols─oxidized cholesterol derivatives, including nuclear-receptor ligands causally linked to skin and neurodegerative diseases, cancer and atherosclerosis. Sulfonation of signaling oxysterols redirects their receptor-binding functions, and controlling these functions is expected to prove valuable in disease prevention and treatment. SULT2B1b is distinct among the human SULT2 isoforms by virtue of its atypically long N-terminus, which extends 15 residues beyond the next longest N-terminus in the family. Here, in silico studies are used to predict that the N-terminal extension forms an allosteric pocket and to identify potential allosteres. One such allostere, quercetin, is used to confirm the existence of the pocket and to demonstrate that allostere binding inhibits turnover. The structure of the pocket is obtained by positioning quercetin on the enzyme, using spin-label-triangulation NMR, followed by NMR distance-constrained molecular dynamics docking. The model is confirmed using a combination of site-directed mutagenesis and initial-rate studies. Stopped-flow ligand-binding studies demonstrate that inhibition is achieved by stabilizing the closed form of the enzyme active-site cap, which encapsulates the nucleotide, slowing its release. Finally, endogenous oxysterols are shown to bind to the site in a highly selective fashion─one of the two immediate biosynthetic precursors of cholesterol (7-dehydrocholesterol) is an inhibitor, while the other (24-dehydrocholesterol) is not. These findings provide insights into the allosteric dialogue in which SULT2B1b participates in and establishes a template against which to develop isoform-specific inhibitors to control SULT2B1b biology.
在人类细胞质磺基转移酶中，SULT2B1b 对氧化甾醇（氧化胆固醇衍生物）具有高度特异性，包括核受体配体，这些配体与皮肤和神经退行性疾病、癌症和动脉粥样硬化有因果关系。信号氧化甾醇的磺化作用改变了它们的受体结合功能，控制这些功能有望在疾病预防和治疗中证明是有价值的。SULT2B1b 与人类 SULT2 同工型不同，因为它的 N 端异常长，比家族中最长的 N 端长 15 个残基。在这里，通过计算机模拟研究来预测 N 端延伸形成别构口袋，并识别潜在的别构体。一种这样的别构体，槲皮素，用于确认口袋的存在并证明别构体结合抑制周转率。通过将槲皮素置于酶上，使用自旋标记三角 NMR 获得口袋的结构，然后进行 NMR 距离约束分子动力学对接。该模型通过结合定点突变和初始速率研究得到证实。停流配体结合研究表明，通过稳定酶活性位点帽的封闭形式来实现抑制，该帽包裹核苷酸，从而减缓其释放。最后，显示内源性氧化甾醇以高度选择性的方式结合到该位点——胆固醇的两个直接生物合成前体之一（7-脱氢胆固醇）是抑制剂，而另一个（24-脱氢胆固醇）则不是。这些发现为 SULT2B1b 参与的别构对话提供了深入的了解，并建立了一个模板，以此来开发同工型特异性抑制剂来控制 SULT2B1b 的生物学功能。
