Turning a protein kinase on or off from a single allosteric site via disulfide trapping
Turning a protein kinase on or off from a single allosteric site via disulfide trapping
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DOI：
10.1073/pnas.1102376108
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发表时间：
2011-04-12
影响因子：
11.1
通讯作者：
Wells, James A.
中科院分区：
文献类型：
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作者：
Sadowsky, Jack D.;Burlingame, Mark A.;Wells, James A.
在酶（如蛋白激酶）中识别和表征变构位点对理解变构机制和药物发现都有很大的兴趣。在这里，我们应用了一种位点定向技术，二硫捕获，从结构和功能上研究了丝氨酸/苏氨酸激酶3-磷酸肌苷依赖性激酶1 (PDK1)- PDK1相互作用片段（PIF）口袋上的变抗位点是如何被下游底物激酶（PIFtides）上的激活肽基序和小分子片段所参与的。通过监测PIFtide和PDK1半胱氨酸突变体之间的成对二硫偶联，我们确定了PIFtide在PDK1 PIF口袋中的结合方向，并评估了PIFtide定位与激酶激活之间的微妙关系。我们还发现了多种小分子片段二硫化物（< 300 Da），它们可以通过偶联PIF口袋激活或抑制PDK1，从而比偶联到相同位点的PIFtides显示出更大的功能多样性。生化数据和三种晶体结构揭示了最佳片段激活剂和抑制剂的作用机制。这些研究表明，二硫捕获对于表征激酶上的变构位点是有用的，并且蛋白激酶上的单个变构位点可以被小分子激活和抑制。
There is significant interest in identifying and characterizing allosteric sites in enzymes such as protein kinases both for understanding allosteric mechanisms as well as for drug discovery. Here, we apply a site-directed technology, disulfide trapping, to interrogate structurally and functionally how an allosteric site on the Ser/Thr kinase, 3-phosphoinositide-dependent kinase 1 (PDK1)-the PDK1-interacting-fragment (PIF) pocket-is engaged by an activating peptide motif on downstream substrate kinases (PIFtides) and by small molecule fragments. By monitoring pairwise disulfide conjugation between PIFtide and PDK1 cysteine mutants, we defined the PIFtide binding orientation in the PIF pocket of PDK1 and assessed subtle relationships between PIFtide positioning and kinase activation. We also discovered a variety of small molecule fragment disulfides (< 300 Da) that could either activate or inhibit PDK1 by conjugation to the PIF pocket, thus displaying greater functional diversity than is displayed by PIFtides conjugated to the same sites. Biochemical data and three crystal structures provided insight into the mechanism of action of the best fragment activators and inhibitors. These studies show that disulfide trapping is useful for characterizing allosteric sites on kinases and that a single allosteric site on a protein kinase can be exploited for both activation and inhibition by small molecules.