Department of Integrative Structural and Computational Biology and Skaggs Institute of Chemical Biology , The Scripps Research Institute , 10550 North Torrey Pines Road , La Jolla , California 92037 , United States.
Biochemistry. 2019 Mar 12;58(10):1354-1362. doi: 10.1021/acs.biochem.8b01290. Epub 2019 Feb 22.
The intrinsically disordered transactivation domains of HIF-1α and CITED2 compete for binding of the TAZ1 domain of the CREB-binding protein by a unidirectional allosteric mechanism involving direct competition for shared binding sites, ternary complex formation, and TAZ1 conformational changes. To gain insight into the mechanism by which CITED2 displaces HIF-1α from TAZ1, we used nuclear magnetic resonance spin relaxation methods to obtain an atomic-level description of the picosecond to nanosecond backbone dynamics that contribute to TAZ1 binding and competition. We show that HIF-1α and CITED2 adopt different dynamics in their complexes with TAZ1, with flexibility observed for HIF-1α in regions that would maintain accessibility for CITED2 to bind to TAZ1 and facilitate subsequent HIF-1α dissociation. In contrast, critical regions of CITED2 adopt a rigid structure in its complex with TAZ1, minimizing the ability of HIF-1α to compete for binding. We also find that TAZ1, previously thought to be a rigid scaffold for binding of disordered protein ligands, displays altered backbone dynamics in its various bound states. TAZ1 is more rigid in its CITED2-bound state than in its free state or in complex with HIF-1α, with increased rigidity observed not only in the CITED2 binding site but also in regions of TAZ1 that undergo conformational changes between the HIF-1α- and CITED2-bound structures. Taken together, these data suggest that backbone dynamics in TAZ1, as well as in the HIF-1α and CITED2 ligands, play a role in modulating the occupancy of TAZ1 and highlight the importance of characterizing both binding partners in molecular interactions.
HIF-1α 和 CITED2 的固有无序转录激活结构域通过一种单向变构机制竞争与 CREB 结合蛋白的 TAZ1 结构域结合，该机制涉及直接竞争共享结合位点、三元复合物形成和 TAZ1 构象变化。为了深入了解 CITED2 如何从 TAZ1 上置换 HIF-1α，我们使用核磁共振自旋弛豫方法获得了有助于 TAZ1 结合和竞争的皮秒到纳秒级的骨架动力学的原子水平描述。我们表明，HIF-1α 和 CITED2 在与 TAZ1 的复合物中采用不同的动力学，在 HIF-1α 中观察到灵活性，这些灵活性可保持 CITED2 与 TAZ1 结合的可及性，并促进随后的 HIF-1α 解离。相比之下，CITED2 中关键区域在其与 TAZ1 的复合物中采用刚性结构，最大限度地减少 HIF-1α 竞争结合的能力。我们还发现，TAZ1 先前被认为是无序蛋白配体结合的刚性支架，在其各种结合状态下显示出改变的骨架动力学。TAZ1 在与 CITED2 结合的状态下比在游离状态或与 HIF-1α 复合的状态下更刚性，不仅在 CITED2 结合位点观察到刚性增加，而且在 TAZ1 的区域观察到刚性增加，这些区域在 HIF-1α 和 CITED2 结合结构之间发生构象变化。总之，这些数据表明 TAZ1 以及 HIF-1α 和 CITED2 配体中的骨架动力学在调节 TAZ1 的占据中起作用，并强调了在分子相互作用中表征两个结合伴侣的重要性。
