Seifi Bahman, Wallin Stefan
Department of Physics and Physical Oceanography, Memorial University of Newfoundland, St Johns, NL, Canada.
Proteins. 2025 Mar;93(3):608-619. doi: 10.1002/prot.26755. Epub 2024 Oct 14.
RfaH is a two-domain metamorphic protein involved in transcription regulation and translation initiation. To carry out its dual functions, RfaH relies on two coupled structural changes: Domain dissociation and fold switching. In the free state, the C-terminal domain (CTD) of RfaH adopts an all-α fold and is tightly associated with the N-terminal domain (NTD). Upon binding to RNA polymerase (RNAP), the domains dissociate and the CTD transforms into an all-β fold while the NTD remains largely, but not entirely, unchanged. We test the idea that a change in the conformation of an extended β-hairpin (β3-β4) located on the NTD, helps trigger domain dissociation. To this end, we use homology modeling to construct a structure, H, which is similar to free RfaH but with a remodeled β3-β4 hairpin. We then use an all-atom physics-based model enhanced with a dual basin structure-based potential to simulate domain separation driven by the thermal unfolding of the CTD with NTD in a fixed, folded conformation. We apply our model to both free RfaH and H. For H we find, in line with our hypothesis, that the CTD exhibits lower stability and the domains dissociate at a lower temperature T, as compared to free RfaH. We do not, however, observe complete refolding to the all-β state in these simulations, suggesting that a change in β3-β4 orientation aids in, but is not sufficient for, domain dissociation. In addition, we study the reverse fold switch in which RfaH returns from a domain-open all-β state to its domain-closed all-α state. We observe a T-dependent transition rate; fold switching is slow at low T, where the CTD tends to be kinetically trapped in its all-β state, and at high-T, where the all-α state becomes unstable. Consequently, our simulations suggest an optimal T at which fold switching is most rapid. At this T, the stabilities of both folds are reduced. Overall, our study suggests that both inter-domain interactions and conformational changes within NTD may be important for the proper functioning of RfaH.
RfaH是一种参与转录调控和翻译起始的双结构域变构蛋白。为了执行其双重功能，RfaH依赖于两种耦合的结构变化：结构域解离和折叠转换。在自由状态下，RfaH的C末端结构域（CTD）采用全α折叠，并与N末端结构域（NTD）紧密结合。与RNA聚合酶（RNAP）结合后，结构域解离，CTD转变为全β折叠，而NTD在很大程度上但并非完全不变。我们测试了位于NTD上的延伸β发夹（β3-β4）构象变化有助于触发结构域解离的想法。为此，我们使用同源建模构建了一个结构H，它与自由RfaH相似，但具有重塑的β3-β4发夹。然后，我们使用基于全原子物理的模型，并通过基于双盆地结构的势进行增强，以模拟在NTD处于固定折叠构象的情况下，CTD的热解折叠驱动的结构域分离。我们将模型应用于自由RfaH和H。对于H，与我们的假设一致，我们发现与自由RfaH相比，CTD表现出更低的稳定性，并且结构域在更低的温度T下解离。然而，在这些模拟中我们没有观察到完全重折叠到全β状态，这表明β3-β4方向的变化有助于但不足以导致结构域解离。此外，我们研究了反向折叠转换，其中RfaH从结构域开放的全β状态返回到其结构域封闭的全α状态。我们观察到一个依赖于温度的转变速率；在低温下折叠转换缓慢，此时CTD在动力学上倾向于被困在其全β状态，而在高温下全α状态变得不稳定。因此，我们的模拟表明存在一个最佳温度T，在该温度下折叠转换最迅速。在这个温度下，两种折叠的稳定性都降低了。总体而言，我们的研究表明结构域间相互作用和NTD内的构象变化对于RfaH的正常功能可能都很重要。
