Enhancing response of a protein conformational switch by using two disordered ligand binding domains.
Enhancing response of a protein conformational switch by using two disordered ligand binding domains.
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通过使用两个无序的配体结合域增强蛋白质构象转换的响应。
DOI：
10.3389/fmolb.2023.1114756
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发表时间：
2023
影响因子：
5
通讯作者：
中科院分区：
文献类型：
--
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简介：蛋白质构象开关通常通过将识别靶配体的输入结构域融合到建立生物反应的输出结构域来构建。先前的设计采用输入结构域的结合诱导折叠来驱动输出结构域的构象变化。添加第二输入结构域原则上可以收获额外的结合能用于执行有用的工作。然而，如何将两个结合结构域融合到单个输出结构域上，使得两个结合结构域的折叠联合收割机组合以实现输出结构域中的构象变化，这并不明显。 研究方法：在这里，我们通过复制其序列的C末端部分并将其附加到其N末端来将核糖核酸酶芽孢杆菌RNA酶（Bn）转化为可切换的酶，从而建立以相互排斥的方式竞争共享核心的天然折叠（OFF状态）和环状排列折叠（ON状态）。将两个拷贝的FK506结合蛋白（FKBP）（均因V24 A突变而变得不稳定，一个已被环状排列）插入到工程化芽孢杆菌RNA酶中共享序列和重复序列之间的连接处。 结果如下：雷帕霉素诱导的FK506结合蛋白的折叠通过互斥折叠效应拉伸和展开barnase的天然折叠，雷帕霉素诱导的FK506结合蛋白的排列折叠通过环闭合熵原理稳定barnase的排列折叠。这些折叠事件相互补充以开启RNA酶功能。细胞毒性转换机制在酵母和人细胞中进行了验证，并在体外用纯化的蛋白质进行验证。 讨论内容：热力学建模和实验结果表明，闭环熵和互斥折叠的双重作用类似于发动机传动装置，其中闭环熵作为低档位，在低配体浓度下提供有效的切换，并且互斥折叠作为高档位，以允许切换在高配体浓度下达到其最大响应。
Introduction: 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. Methods: 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. Results: Rapamycin-induced folding of FK506 binding protein stretched and unfolded the native fold of barnase via 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 in vitro with purified protein. Discussion: 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.
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通讯作者：
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