Allosteric cooperation in a de novo-designed two-domain protein.
Allosteric cooperation in a de novo-designed two-domain protein.
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DOI：
10.1073/pnas.2017062117
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发表时间：
2020-12-29
影响因子：
11.1
通讯作者：
DeGrado WF
中科院分区：
文献类型：
--
作者：
Pirro F;Schmidt N;Lincoff J;Widel ZX;Polizzi NF;Liu L;Therien MJ;Grabe M;Chino M;Lombardi A;DeGrado WF
进化的一个主要机制涉及融合编码单结构域蛋白质的基因，以创建实现新功能的多结构域结构。在这里，我们开发的方法来设计多结构域蛋白质完全从头开始，并实现了一个变构调节的酚氧化酶，响应合成卟啉的结合总理从头设计。我们描述了从头设计的变构调节蛋白，它包括两个紧密耦合的结构域。一个结构域基于DF（意大利语中的Due Ferri或英语中的two-iron）从头蛋白家族，其具有催化酚氧化酶反应的二铁辅因子，而第二个结构域基于PS1（卟啉结合序列），其结合合成的锌卟啉（ZnP）。ZnP与原始PS1蛋白的结合引起结构和动力学的变化，我们预期这会影响融合DF结构域的催化速率。DF和PS1都是四螺旋束，但它们具有不同的束结构。为了实现结构域之间的紧密耦合，使用计算方法将它们通过四个螺旋连接体连接，以发现能够跨越两种架构的最可设计的连接。得到的蛋白质DFP 1（Due Ferri Porphyrin）以预期的方式结合两个辅因子。完全重构的DFP 1的晶体结构也与设计非常一致，并且它显示ZnP辅因子与双金属中心结合超过12 μ m。接下来，将导致二铁中心的底物结合裂缝引入DFP 1。所产生的蛋白质充当变构调节的酚氧化酶。它的Michaelis-Menten参数的强烈影响，通过结合的ZnP，导致四倍更紧的Km和7倍的kcat下降。这些研究建立了设计变构调节催化蛋白的可行性，完全从头开始。
A major mechanism of evolution involves fusing genes that encode single-domain proteins to create multidomain structures that achieve new functions. Here, we develop methods to design multidomain proteins entirely from scratch and achieve the premier de novo design of an allosterically regulated phenol oxidase that responds to the binding of a synthetic porphyrin. We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis–Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter Km and a 7-fold decrease in kcat. These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.
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影响因子：
16.1
作者：
Chen, Xiaoying;Zaro, Jennica L.;Shen, Wei-Chiang
通讯作者：
Shen, Wei-Chiang
影响因子：
16.6
作者：
Chino, Marco;Leone, Linda;Lombardi, Angela
通讯作者：
Lombardi, Angela
影响因子：
5.6
作者：
Grigoryan G;Degrado WF
通讯作者：
Degrado WF
影响因子：
2.2
作者：
de la Torre, JG;Huertas, ML;Carrasco, B
通讯作者：
Carrasco, B
DOI：
10.1039/j29690000823
发表时间：
1969-01-01
期刊：
JOURNAL OF THE CHEMICAL SOCIETY B-PHYSICAL ORGANIC
影响因子：
--
作者：
CORBETT, JF
通讯作者：
CORBETT, JF