Fast Association and Slow Transitions in the Interaction between Two Intrinsically Disordered Protein Domains
Fast Association and Slow Transitions in the Interaction between Two Intrinsically Disordered Protein Domains
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DOI：
10.1074/jbc.m112.399436
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发表时间：
2012-10-05
影响因子：
4.8
通讯作者：
Jemth, Per
中科院分区：
文献类型：
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作者：
Dogan, Jakob;Schmidt, Tanja;Jemth, Per
含有长无序区域的蛋白质在蛋白质组中普遍存在，并且经常与疾病相关。然而，这种内在无序蛋白（IDP）识别其靶标的机制还不清楚。在这里，我们报告的第一个实验研究的相互作用动力学的核共激活剂结合结构域的CREB结合蛋白和激活结构域的p160转录共激活甲状腺激素和类维生素A受体。两个蛋白质结构域在自由状态下本质上是无序的，并且在彼此结合时协同折叠。利用停流技术，我们发现结合反应是快速的，在277 K下的缔合速率常数为3 × 10（7）M-1 s（-1）。一个保守的埋藏的分子间盐桥的突变表明，静电支配的快速协会。此外，在盐桥突变或在高盐浓度下，检测到额外的动力学阶段（在277 K下分别类似于20和类似于40 s（-1）），表明盐桥可以在分子内步骤中引导生产性双分子复合物的形成。最后，我们直接测量了与结合后的构象转变相关的IDP结构域的缓慢动力学（类似于277 K时的1 s（-1））。总之，实验表明相互作用涉及几个步骤和中间状态的积累。我们的数据是一致的诱导适合机制，与以前的模拟。我们建议，缓慢的过渡可能是国内流离失所者的多伙伴互动的结果。
Proteins that contain long disordered regions are prevalent in the proteome and frequently associated with diseases. However, the mechanisms by which such intrinsically disordered proteins (IDPs) recognize their targets are not well understood. Here, we report the first experimental investigation of the interaction kinetics of the nuclear co-activator binding domain of CREB-binding protein and the activation domain from the p160 transcriptional co-activator for thyroid hormone and retinoid receptors. Both protein domains are intrinsically disordered in the free state and synergistically fold upon binding each other. Using the stopped-flow technique, we found that the binding reaction is fast, with an association rate constant of 3 x 10(7) M-1 s(-1) at 277 K. Mutation of a conserved buried intermolecular salt bridge showed that electrostatics govern the rapid association. Furthermore, upon mutation of the salt bridge or at high salt concentration, an additional kinetic phase was detected (similar to 20 and similar to 40 s(-1), respectively, at 277 K), suggesting that the salt bridge may steer formation of the productive bimolecular complex in an intramolecular step. Finally, we directly measured slow kinetics for the IDP domains (similar to 1 s(-1) at 277 K) related to conformational transitions upon binding. Together, the experiments demonstrate that the interaction involves several steps and accumulation of intermediate states. Our data are consistent with an induced fit mechanism, in agreement with previous simulations. We propose that the slow transitions may be a consequence of the multipartner interactions of IDPs.