A method to trap transient and weak interacting protein complexes for structural studies.
A method to trap transient and weak interacting protein complexes for structural studies.
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DOI：
10.4161/idp.25464
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发表时间：
2013-01
期刊：
影响因子：
--
通讯作者：
Sivaraman J
中科院分区：
文献类型：
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作者：
Chichili VPR;Kumar V;Sivaraman J
几个关键的生物事件在结合伙伴之间的瞬时相互作用中采用了“打了就跑”的策略。在某些情况下，其中一个结合伙伴的无序性质严重阻碍了共结晶的成功，往往只导致其中一个伙伴的结晶。在这里，我们讨论了一种捕获弱的和瞬时的蛋白质相互作用以进行结晶的方法。这种方法需要至少一个相互作用伙伴的结构细节和结合知识，以使用最佳大小的接头将蛋白质的已知最小结合区域(肽)对接到另一个上。在结晶之前，应验证纯化的连接结构的完整折叠和稳定性。在结构确定之后，对独立的、全长的未连接的蛋白质进行结构引导的功能研究，以验证连接的复合体的发现。我们设计了这种方法，然后使用神经元特异性底物神经颗粒素和神经调制素的24个氨基酸的最小结合区域来验证其有效性，这些底物通过甘氨酸连接物连接到它们相互作用的伙伴钙调蛋白。此外，报道的对独立全长蛋白质的功能研究证实了连接的多肽复合体的发现。基于我们的研究，并结合支持文献，我们认为优化的连接子可以提供一个环境来模拟结合伙伴之间的自然相互作用，并为结构研究提供一种有用的策略，以捕获涉及几个生物过程的弱和瞬时相互作用。
Several key biological events adopt a “hit-and-run” strategy in their transient interactions between binding partners. In some instances, the disordered nature of one of the binding partners severely hampers the success of co-crystallization, often leading to the crystallization of just one of the partners. Here, we discuss a method to trap weak and transient protein interactions for crystallization. This approach requires the structural details of at least one of the interacting partners and binding knowledge to dock the known minimum binding region (peptide) of the protein onto the other using an optimal-sized linker. Prior to crystallization, the purified linked construct should be verified for its intact folding and stability. Following structure determination, structure-guided functional studies are performed with independent, full-length unlinked proteins to validate the findings of the linked complex. We designed this approach and then validated its efficacy using a 24 amino acid minimum binding region of the intrinsically disordered, neuron-specific substrates, Neurogranin and Neuromodulin, joined via a Gly-linker to their interacting partner, Calmodulin. Moreover, the reported functional studies with independent full-length proteins confirmed the findings of the linked peptide complexes. Based on our studies, and in combination with the supporting literature, we suggest that optimized linkers can provide an environment to mimic the natural interactions between binding partners, and offer a useful strategy for structural studies to trap weak and transient interactions involved in several biological processes.