The designability of protein switches by chemical rescue of structure: mechanisms of inactivation and reactivation.
The designability of protein switches by chemical rescue of structure: mechanisms of inactivation and reactivation.
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通过结构的化学拯救来设计蛋白质开关：失活和再激活的机制。
DOI：
10.1021/ja407644b
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发表时间：
2013
影响因子：
15
通讯作者：
Karanicolas,John
中科院分区：
文献类型：
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作者：
Xia,Yan;DiPrimio,Nina;Keppel,TheodoreR;Vo,Binh;Fraser,Keith;Battaile,KevinP;Egan,Chet;Bystroff,Christopher;Lovell,Scott;Weis,DavidD;Anderson,JChristopher;Karanicolas,John
通过药理学试剂选择性激活特定蛋白质功能的能力是化学生物学的长期目标。最近，我们报道了一种直接在酶的催化结构域中设计新变构效应位点的方法。这种方法与传统的酶的化学拯救不同，因为它依赖于结构的破坏和恢复，而不是活性位点化学，作为实现调节功能的手段。然而，合理地确定类似的nobinding网站在其他酶是一个关键的挑战，扩展这种方法引入变构控制到其他酶。在这里，我们表明，突变位点导致蛋白质失活，通过色氨酸甘氨酸取代，并允许（部分）重新激活，随后加入吲哚是非常频繁的。通过一系列的方法，包括基于细胞的报告分析，计算结构预测和能量分析，荧光研究，酶学，脉冲蛋白水解，X射线晶体学和氢氘质谱，我们发现这些可转换的蛋白质最常见的是通过控制蛋白质的稳定性间接调节。在这些情况下，吲哚的加入不是通过恢复离散的构象变化来挽救活性，正如我们在先前报道的唯一实施例中所观察到的那样，而是通过恢复蛋白质稳定性来挽救活性。这一重要发现将极大地影响未来开关和传感器的设计，因为评估与空腔形成突变相关的稳定性差异比预测变构构象变化更容易处理。通过类比天然信号系统，本研究的见解进一步提高了调节稳定性的令人兴奋的前景，以将最佳识别特性设计到未来的novoswitches和传感器中，通过结构的化学拯救构建。
The ability to selectively activate function of particular proteins via pharmacological agents is a longstanding goal in chemical biology. Recently, we reported an approach for designing ade novoallosteric effector site directly into the catalytic domain of an enzyme. This approach is distinct from traditional chemical rescue of enzymes in that it relies on disruption and restoration of structure, rather than active site chemistry, as a means to achieve modulate function. However, rationally identifying analogousde novobinding sites in other enzymes represents a key challenge for extending this approach to introduce allosteric control into other enzymes. Here we show that mutation sites leading to protein inactivation via tryptophan-to-glycine substitution and allowing (partial) reactivation by the subsequent addition of indole are remarkably frequent. Through a suite of methods including a cell-based reporter assay, computational structure prediction and energetic analysis, fluorescence studies, enzymology, pulse proteolysis, X-ray crystallography, and hydrogen–deuterium mass spectrometry, we find that these switchable proteins are most commonly modulatedindirectly, through control of protein stability. Addition of indole in these cases rescues activity not by reverting a discrete conformational change, as we had observed in the sole previously reported example, but rather rescues activity by restoring protein stability. This important finding will dramatically impact the design of future switches and sensors built by this approach, since evaluating stability differences associated with cavity-forming mutations is a far more tractable task than predicting allosteric conformational changes. By analogy to natural signaling systems, the insights from this study further raise the exciting prospect of modulating stability to design optimal recognition properties into futurede novoswitches and sensors built through chemical rescue of structure.