A magnesium-induced triplex pre-organizes the SAM-II riboswitch.
A magnesium-induced triplex pre-organizes the SAM-II riboswitch.
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DOI：
10.1371/journal.pcbi.1005406
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发表时间：
2017-03
影响因子：
4.3
通讯作者：
Sanbonmatsu KY
中科院分区：
文献类型：
--
作者：
Roy S;Lammert H;Hayes RL;Chen B;LeBlanc R;Dayie TK;Onuchic JN;Sanbonmatsu KY
我们的13 C-和1H-化学交换饱和转移（CEST）实验先前揭示了在配体的情况下，SAM-II核糖开关的部分闭合和开放构象之间的动态交换。在这里，所有原子结构为基础的分子模拟，与曼宁反离子凝聚和明确的镁离子的静电效应计算的SAM-II核糖开关的折叠自由能景观。我们使用这种分析来预测镁离子重塑景观，将平衡从延伸的，部分未折叠的状态转移到紧凑的，预组织的构象，类似于配体结合状态。我们的CEST和SAXS实验，在不同的镁离子浓度，定量地证实了我们的模拟结果，表明镁离子诱导崩溃和预组织。理论与实验之间的一致性支持了我们模拟的微观解释，这表明螺旋P2b和环L1之间的三链体形成对镁高度敏感，并在预组织中起着关键作用。SAM-II核糖开关的预组织允许以高选择性快速检测配体，这对于生物功能是重要的。带正电荷的金属离子的存在对于维持RNA的结构折叠和功能至关重要。在不同的金属离子中，镁对RNA的稳定性特别重要，因为它可以有效地支持RNA折叠中带负电荷的磷酸基团的紧密组装。SAM-II核糖开关是经典假结折叠的一个例子，其结合S-腺苷甲硫氨酸，稳定替代折叠形式以抑制基因表达。在我们早期的13 C-和1H-化学交换饱和转移（CEST）实验中，我们发现了一个次要的，部分封闭和主要的，开放状态的构象在配体的情况下，构象之间的转变。我们在不同镁浓度下的CEST实验现在表明，镁离子可以诱导apo SAM-II核糖开关中的构象预组织，这有望促进配体结合。为了理解这种镁诱导的转变的微观细节，我们进行了基于全原子结构的分子模拟，包括静电和明确的镁离子。我们的自由能计算表明，部分封闭的预组织状态进一步稳定与镁浓度的增加。这与我们的13 C-CEST配置文件，SAXS和尺寸排阻色谱数据，以及最近的单分子FRET实验非常一致。我们的研究结果表明，足够高浓度的镁是必不可少的预组织载脂蛋白SAM-II核糖开关。
Our 13C- and 1H-chemical exchange saturation transfer (CEST) experiments previously revealed a dynamic exchange between partially closed and open conformations of the SAM-II riboswitch in the absence of ligand. Here, all-atom structure-based molecular simulations, with the electrostatic effects of Manning counter-ion condensation and explicit magnesium ions are employed to calculate the folding free energy landscape of the SAM-II riboswitch. We use this analysis to predict that magnesium ions remodel the landscape, shifting the equilibrium away from the extended, partially unfolded state towards a compact, pre-organized conformation that resembles the ligand-bound state. Our CEST and SAXS experiments, at different magnesium ion concentrations, quantitatively confirm our simulation results, demonstrating that magnesium ions induce collapse and pre-organization. Agreement between theory and experiment bolsters microscopic interpretation of our simulations, which shows that triplex formation between helix P2b and loop L1 is highly sensitive to magnesium and plays a key role in pre-organization. Pre-organization of the SAM-II riboswitch allows rapid detection of ligand with high selectivity, which is important for biological function. The presence of positively charged metal ions is essential to maintain the structural fold and function of RNA. Among different metal ions, magnesium is particularly important for the stability of RNA because it can efficiently support a close assembly of negatively charged phosphate groups in an RNA fold. The SAM-II riboswitch is an example of a classical pseudoknot fold, which binds S-adenosyl methionine, stabilizing an alternate folded form to inhibit gene expression. In our early 13C- and 1H-chemical exchange saturation transfer (CEST) experiments, we found a conformational transition between a minor, partially closed and a major, open state conformation in the absence of ligand. Our CEST experiments at different magnesium concentrations now suggest that magnesium ions can induce a conformational pre-organization in the apo SAM-II riboswitch, which is expected to facilitate ligand binding. To understand the microscopic details of this magnesium-induced transition, we perform all-atom structure-based molecular simulations including electrostatics and explicit magnesium ions. Our free energy calculations reveal that the partially closed pre-organized state is further stabilized with increasing magnesium concentration. This is in excellent agreement with our 13C-CEST profile, SAXS, and size-exclusion chromatographic data, and with recent single molecule FRET experiments. Our results suggest that a sufficiently high concentration of magnesium is essential to pre-organize the apo SAM-II riboswitch.