Residual Structures, Conformational Fluctuations, and Electrostatic Interactions in the Synergistic Folding of Two Intrinsically Disordered Proteins

To understand the interplay of residual structures and conformational fluctuations in the interaction of intrinsically disordered proteins (IDPs), we first combined implicit solvent and replica exchange sampling to calculate atomistic disordered ensembles of the nuclear co-activator binding domain (NCBD) of transcription coactivator CBP and the activation domain of the p160 steroid receptor coactivator ACTR. The calculated ensembles are in quantitative agreement with NMR-derived residue helicity and recapitulate the experimental observation that, while free ACTR largely lacks residual secondary structures, free NCBD is a molten globule with a helical content similar to that in the folded complex. Detailed conformational analysis reveals that free NCBD has an inherent ability to substantially sample all the helix configurations that have been previously observed either unbound or in complexes. Intriguingly, further high-temperature unbinding and unfolding simulations in implicit and explicit solvents emphasize the importance of conformational fluctuations in synergistic folding of NCBD with ACTR. A balance between preformed elements and conformational fluctuations appears necessary to allow NCBD to interact with different targets and fold into alternative conformations. Together with previous topology-based modeling and existing experimental data, the current simulations strongly support an “extended conformational selection” synergistic folding mechanism that involves a key intermediate state stabilized by interaction between the C-terminal helices of NCBD and ACTR. In addition, the atomistic simulations reveal the role of long-range as well as short-range electrostatic interactions in cooperating with readily fluctuating residual structures, which might enhance the encounter rate and promote efficient folding upon encounter for facile binding and folding interactions of IDPs. Thus, the current study not only provides a consistent mechanistic understanding of the NCBD/ACTR interaction, but also helps establish a multi-scale molecular modeling framework for understanding the structure, interaction, and regulation of IDPs in general.为阐明内在无序蛋白（intrinsically disordered proteins, IDPs）相互作用中残基结构与构象波动的协同机制，本研究首先结合隐式溶剂模型与副本交换采样方法，计算得到转录辅激活因子CBP的核辅激活因子结合结构域（nuclear co-activator binding domain, NCBD）以及p160类固醇受体辅激活因子ACTR的激活结构域的全原子无序系综。计算得到的系综与核磁共振（nuclear magnetic resonance, NMR）测得的残基螺旋倾向性定量吻合，且重现了实验观测结果：游离态ACTR几乎不存在残留二级结构，而游离态NCBD为熔球态，其螺旋含量与折叠复合物中的螺旋含量相近。详细的构象分析表明，游离态NCBD天生具备充分采样所有曾在游离态或复合物态中观测到的螺旋构象的能力。有趣的是，在隐式与显式溶剂模型中开展的进一步高温解折叠与解离模拟，凸显了构象波动在NCBD与ACTR协同折叠过程中的关键作用。预形成结构单元与构象波动之间的动态平衡，似乎是NCBD能够与不同靶标结合并折叠为可变构象的必要条件。结合此前基于拓扑的建模研究与现有实验数据，本研究的模拟结果有力支持了"扩展构象选择"协同折叠机制：该机制中存在一个由NCBD与ACTR的C端螺旋相互作用稳定的关键中间态。此外，全原子模拟还揭示了长程与短程静电相互作用与易波动的残留结构协同发挥的作用：这一协同作用可提升IDP结合过程中的遭遇速率，并在遭遇后促进高效折叠，从而助力IDP的高效结合与折叠相互作用。综上，本研究不仅为NCBD与ACTR的相互作用提供了一致的机制性理解，还为构建通用的IDP结构、相互作用与调控机制的多尺度分子建模框架奠定了基础。