Energetics and kinetics of substrate analog-coupled staphylococcal nuclease folding revealed by a statistical mechanical approach.
Energetics and kinetics of substrate analog-coupled staphylococcal nuclease folding revealed by a statistical mechanical approach.
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通过统计机械方法揭示底物模拟耦合葡萄球菌核酸酶折叠的能量学和动力学。
DOI：
10.1073/pnas.1914349117
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发表时间：
2020
影响因子：
11.1
通讯作者：
Maki,Kosuke
中科院分区：
文献类型：
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作者：
Mizukami,Takuya;Furuzawa,Shunta;Itoh,SatoruG;Segawa,Saho;Ikura,Teikichi;Ihara,Kunio;Okumura,Hisashi;Roder,Heinrich;Maki,Kosuke
与配体结合相关的蛋白质构象变化，特别是那些涉及内在无序的蛋白质，是由紧密耦合的分子内和分子间事件介导的。这种反应通常在两个限制动力学机制方面进行讨论，构象选择（CS），其中折叠先于结合，和诱导拟合（IF），其中结合先于折叠。已经表明，偶联的折叠/结合反应可以沿着CS和IF途径进行，其通量比取决于配体浓度等条件。然而，这种复杂反应的结构和能量基础仍然知之甚少。因此，我们使用实验，理论和计算方法来探索葡萄球菌核酸酶在底物类似物腺苷-3 ′，5 ′-二磷酸存在下的偶联折叠/结合反应的结构和能量方面。光学监测的平衡和动力学数据，结合统计力学模型，更深入地了解特定的和库仑蛋白质-配体相互作用的相对重要性，在管理的反应机制。我们还研究了结构方面的反应，在残留物水平上使用NMR和全原子复制置换分子动力学模拟。这两种方法都产生了明确的证据积累的瞬时蛋白质-配体遇到复杂的IF-显性条件下的反应早期。平衡/动力学折叠的定量分析表明，配体依赖的CS-IF移位导致稳定的紧凑的过渡态主要是由弱配体依赖的库仑相互作用与较小的贡献，从特定的结合能。在更宏观的层面上，CS到IF的位移表示为自由能表面上反应“路线”的位移，这与通量分析一致。
Protein conformational changes associated with ligand binding, especially those involving intrinsically disordered proteins, are mediated by tightly coupled intra- and intermolecular events. Such reactions are often discussed in terms of two limiting kinetic mechanisms, conformational selection (CS), where folding precedes binding, and induced fit (IF), where binding precedes folding. It has been shown that coupled folding/binding reactions can proceed along both CS and IF pathways with the flux ratio depending on conditions such as ligand concentration. However, the structural and energetic basis of such complex reactions remains poorly understood. Therefore, we used experimental, theoretical, and computational approaches to explore structural and energetic aspects of the coupled-folding/binding reaction of staphylococcal nuclease in the presence of the substrate analog adenosine-3′,5′-diphosphate. Optically monitored equilibrium and kinetic data, combined with a statistical mechanical model, gave deeper insight into the relative importance of specific and Coulombic protein–ligand interactions in governing the reaction mechanism. We also investigated structural aspects of the reaction at the residue level using NMR and all-atom replica-permutation molecular dynamics simulations. Both approaches yielded clear evidence for accumulation of a transient protein–ligand encounter complex early in the reaction under IF-dominant conditions. Quantitative analysis of the equilibrium/kinetic folding revealed that the ligand-dependent CS-to-IF shift resulted from stabilization of the compact transition state primarily by weakly ligand-dependent Coulombic interactions with smaller contributions from specific binding energies. At a more macroscopic level, the CS-to-IF shift was represented as a displacement of the reaction “route” on the free energy surface, which was consistent with a flux analysis.