Research Center of Integrative Molecular Systems (CIMoS), Institute for Molecular Science (IMS), National Institutes of Natural Sciences (NINS), Okazaki, Japan.
Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences (NINS), Okazaki, Japan.
Nat Chem. 2023 Nov;15(11):1591-1598. doi: 10.1038/s41557-023-01256-4. Epub 2023 Jul 6.
Allostery produces concerted functions of protein complexes by orchestrating the cooperative work between the constituent subunits. Here we describe an approach to create artificial allosteric sites in protein complexes. Certain protein complexes contain subunits with pseudo-active sites, which are believed to have lost functions during evolution. Our hypothesis is that allosteric sites in such protein complexes can be created by restoring the lost functions of pseudo-active sites. We used computational design to restore the lost ATP-binding ability of the pseudo-active site in the B subunit of a rotary molecular motor, V-ATPase. Single-molecule experiments with X-ray crystallography analyses revealed that binding of ATP to the designed allosteric site boosts this V's activity compared with the wild-type, and the rotation rate can be tuned by modulating ATP's binding affinity. Pseudo-active sites are widespread in nature, and our approach shows promise as a means of programming allosteric control over concerted functions of protein complexes.
变构作用通过协调组成亚基之间的协作来产生蛋白质复合物的协同功能。在这里，我们描述了一种在蛋白质复合物中创建人工变构位点的方法。某些蛋白质复合物包含具有伪活性位点的亚基，据信这些亚基在进化过程中丧失了功能。我们的假设是，通过恢复伪活性位点的丢失功能，可以在这类蛋白质复合物中创建变构位点。我们使用计算设计来恢复旋转分子马达 V-ATPase B 亚基中伪活性位点的丢失 ATP 结合能力。利用 X 射线晶体学分析的单分子实验表明，与野生型相比，设计的变构位点与 ATP 的结合可增强 V 的活性，并且可以通过调节 ATP 的结合亲和力来调整旋转速率。伪活性位点在自然界中广泛存在，我们的方法为编程对蛋白质复合物协同功能的变构控制提供了一种有前途的手段。
