Rational design of a ligand-controlled protein conformational switch
Rational design of a ligand-controlled protein conformational switch
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DOI：
10.1073/pnas.1218319110
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发表时间：
2013-04-23
影响因子：
11.1
通讯作者：
Dokholyan, Nikolay V.
中科院分区：
文献类型：
--
作者：
Dagliyan, Onur;Shirvanyants, David;Dokholyan, Nikolay V.
设计可调节的多态蛋白是蛋白质工程的一个挑战。在这里，我们设计了一种具有独特拓扑结构的蛋白质，称为uniRapR，其构象由小分子的结合控制。我们在硅、体外和体内证实了uniRapR的开关和控制能力。作为概念的证明，uniRapR被用作人工调节结构域来控制激酶的活性。通过在单个细胞和整个生物体中使用uniRapR激活Src激酶，我们观察到两种独特的表型与其在转移中的作用一致。Src激酶的激活导致斑马鱼表皮组织中HeLa细胞极化扩散的突起和细胞间接触缺失的形态学改变。uniRapR的合理创建体现了计算蛋白设计的力量，并为靶向激活许多途径以研究生物体中的信号传导提供了强有力的手段。
Design of a regulatable multistate protein is a challenge for protein engineering. Here we design a protein with a unique topology, called uniRapR, whose conformation is controlled by the binding of a small molecule. We confirm switching and control ability of uniRapR in silico, in vitro, and in vivo. As a proof of concept, uniRapR is used as an artificial regulatory domain to control activity of kinases. By activating Src kinase using uniRapR in single cells and whole organism, we observe two unique phenotypes consistent with its role in metastasis. Activation of Src kinase leads to rapid induction of protrusion with polarized spreading in HeLa cells, and morphological changes with loss of cell-cell contacts in the epidermal tissue of zebrafish. The rational creation of uniRapR exemplifies the strength of computational protein design, and offers a powerful means for targeted activation of many pathways to study signaling in living organisms.