Furuya Akihiro, Kawano Fuun, Nakajima Takahiro, Ueda Yoshibumi, Sato Moritoshi
Graduate School of Arts and Sciences, The University of Tokyo , 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
AMED-PRIME, Japan Agency for Medical Research and Development , Tokyo, Japan.
ACS Synth Biol. 2017 Jun 16;6(6):1086-1095. doi: 10.1021/acssynbio.7b00022. Epub 2017 Mar 3.
We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein-protein interactions and genome editing, although the system could be improved further. To enhance the ability of pMagFast2 (a pMag variant with fast kinetics) to bind nMag, we introduced several pMagFast2 modules in tandem into a single construct, pMagFast2(3×). However, the expression level of this construct decreased drastically with increasing number of pMagFast2 molecules integrated into a single construct. In the present study, we applied a new approach to improve the Magnet system based on an assembly domain (AD). Among several ADs, the Ca/calmodulin-dependent protein kinase IIα association domain (CAD) most enhanced the Magnet system. The present CAD-Magnet system overcame a trade-off issue between the expression level and binding affinity. The CAD-converged 12 pMag photoswitches exhibited a stronger interaction with nMag after blue light irradiation compared with monomeric pMag. Additionally, the CAD played a key role in converging effector proteins as well in a single complex. Owing to these substantial improvements, the CAD-Magnet system combined with Tiam1 allowed us to robustly induce localized formation of vertical ruffles on the apical plasma membrane. The CAD-Magnet system combined with 4D imaging was instrumental in revealing the dynamics of ruffle formation.
我们之前开发了磁体系统，它由两种不同的Vivid蛋白变体组成，一种带正电荷，一种带负电荷，分别命名为正磁体（pMag）和负磁体（nMag）。这两种蛋白通过静电相互作用彼此结合，防止不必要的同二聚化，并提供选择性光诱导异二聚化。磁体系统能够操纵细胞功能，如蛋白质-蛋白质相互作用和基因组编辑，尽管该系统还可以进一步改进。为了增强pMagFast2（一种具有快速动力学的pMag变体）与nMag结合的能力，我们将几个pMagFast2模块串联引入到一个单一构建体pMagFast2(3×)中。然而，随着整合到单个构建体中的pMagFast2分子数量增加，该构建体的表达水平急剧下降。在本研究中，我们应用了一种基于组装结构域（AD）的新方法来改进磁体系统。在几种AD中，钙/钙调蛋白依赖性蛋白激酶IIα结合结构域（CAD）对磁体系统的增强作用最为显著。目前的CAD-磁体系统克服了表达水平和结合亲和力之间的权衡问题。与单体pMag相比，CAD融合的12个pMag光开关在蓝光照射后与nMag表现出更强的相互作用。此外，CAD在将效应蛋白汇聚到单个复合物中也起着关键作用。由于这些实质性的改进，与Tiam1结合的CAD-磁体系统使我们能够在顶端质膜上稳健地诱导垂直微褶的局部形成。与4D成像结合的CAD-磁体系统有助于揭示微褶形成的动力学。
