Sekhon Harsimranjit, Ha Jeung-Hoi, Loh Stewart N
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, United States.
Front Mol Biosci. 2023 Mar 2;10:1114756. doi: 10.3389/fmolb.2023.1114756. eCollection 2023.
Protein conformational switches are often constructed by fusing an input domain, which recognizes a target ligand, to an output domain that establishes a biological response. Prior designs have employed binding-induced folding of the input domain to drive a conformational change in the output domain. Adding a second input domain can in principle harvest additional binding energy for performing useful work. It is not obvious, however, how to fuse two binding domains to a single output domain such that folding of both binding domains combine to effect conformational change in the output domain. Here, we converted the ribonuclease barnase (Bn) to a switchable enzyme by duplicating a C-terminal portion of its sequence and appending it to its N-terminus, thereby establishing a native fold (OFF state) and a circularly permuted fold (ON state) that competed for the shared core in a mutually exclusive fashion. Two copies of FK506 binding protein (FKBP), both made unstable by the V24A mutation and one that had been circularly permuted, were inserted into the engineered barnase at the junctions between the shared and duplicated sequences. Rapamycin-induced folding of FK506 binding protein stretched and unfolded the native fold of barnase the mutually exclusive folding effect, and rapamycin-induced folding of permuted FK506 binding protein stabilized the permuted fold of barnase by the loop-closure entropy principle. These folding events complemented each other to turn on RNase function. The cytotoxic switching mechanism was validated in yeast and human cells, and with purified protein. Thermodynamic modeling and experimental results revealed that the dual action of loop-closure entropy and mutually exclusive folding is analogous to an engine transmission in which loop-closure entropy acts as the low gear, providing efficient switching at low ligand concentrations, and mutually exclusive folding acts as the high gear to allow the switch to reach its maximum response at high ligand concentrations.
蛋白质构象开关通常通过将识别目标配体的输入结构域与建立生物反应的输出结构域融合来构建。先前的设计采用结合诱导的输入结构域折叠来驱动输出结构域的构象变化。原则上，添加第二个输入结构域可以获取额外的结合能以执行有用的工作。然而，如何将两个结合结构域融合到单个输出结构域，使得两个结合结构域的折叠共同作用以实现输出结构域的构象变化，这并不明显。在这里，我们通过复制核糖核酸酶巴那斯酶（Bn）序列的C末端部分并将其附加到其N末端，将其转化为可切换的酶，从而建立了一种天然折叠（关闭状态）和一种环状排列折叠（开启状态），它们以互斥的方式竞争共享核心。两个FK506结合蛋白（FKBP）拷贝，均通过V24A突变使其不稳定，其中一个进行了环状排列，被插入到工程化的巴那斯酶中共享序列和重复序列之间的连接处。雷帕霉素诱导的FK506结合蛋白折叠拉伸并展开了巴那斯酶的天然折叠——互斥折叠效应，而雷帕霉素诱导的环状排列的FK506结合蛋白折叠通过环闭合熵原理稳定了巴那斯酶的环状排列折叠。这些折叠事件相互补充以开启核糖核酸酶功能。细胞毒性切换机制在酵母和人类细胞中以及使用纯化蛋白进行了验证。热力学建模和实验结果表明，环闭合熵和互斥折叠的双重作用类似于发动机变速器，其中环闭合熵充当低速档，在低配体浓度下提供高效切换，而互斥折叠充当高速档，使开关在高配体浓度下达到最大响应。
