Death Receptor 5 Activation Is Energetically Coupled to Opening of the Transmembrane Domain Dimer.
Death Receptor 5 Activation Is Energetically Coupled to Opening of the Transmembrane Domain Dimer.
复制标题
死亡受体 5 的激活与跨膜域二聚体的打开呈能量耦合。
DOI：
10.1016/j.bpj.2017.05.038
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发表时间：
2017
影响因子：
3.4
通讯作者：
Sachs,JonathanN
中科院分区：
文献类型：
--
作者：
Vunnam,Nagamani;Campbell-Bezat,CecilyKristine;Lewis,AndrewK;Sachs,JonathanN
肿瘤坏死因子配体与其相应受体的细胞外结构域的结合通过质膜传递信号的精确机制仍然是难以捉摸的。最近的研究提出，包括死亡受体5在内的几种肿瘤坏死因子受体的激活涉及二硫键连接的跨膜（TM）二聚体的剪刀状打开。使用时间分辨荧光共振能量转移，我们提供，据我们所知，第一个直接的生物物理证据，死亡受体5 TM-二聚体开放响应配体结合。然后，为了探测在受体活化的整体能量学中封闭到开放的TM结构域转变的重要性，我们在预测的紧密堆积的TM结构域二聚体界面中设计了点突变体（丙氨酸到苯丙氨酸）。我们假设，庞大的残基应该不稳定的封闭构象和消除的103千卡/摩尔的能量障碍TM域开放和102千卡/摩尔的能量差之间的封闭和开放状态，从而过度敏感的受体。为了测试这一点，我们使用了全原子分子动力学模拟的孤立TM域在明确的脂质双层耦合到平均力计算的热力学势。我们发现，界面上的单点突变体如预测的那样改变了能量格局，但不足以完全消除开放的障碍。然而，计算模型确实预测了在TM结构域二聚体的中心处的双突变ati，i+4位置消除了屏障并且相对于闭合构象稳定了开放构象。我们测试了这些突变体在细胞中的时间分辨荧光共振能量转移和死亡测定，并显示出显着的协议与计算。单突变体对TM结构域分离和细胞死亡的影响很小，而双突变体显著增加了TM结构域分离，并使细胞对配体刺激的敏感性增加了一倍以上。
The precise mechanism by which binding of tumor necrosis factor ligands to the extracellular domain of their corresponding receptors transmits signals across the plasma membrane has remained elusive. Recent studies have proposed that activation of several tumor necrosis factor receptors, including Death Receptor 5, involves a scissorlike opening of the disulfide-linked transmembrane (TM) dimer. Using time-resolved fluorescence resonance energy transfer, we provide, to our knowledge, the first direct biophysical evidence that Death Receptor 5 TM-dimers open in response to ligand binding. Then, to probe the importance of the closed-to-open TM domain transition in the overall energetics of receptor activation, we designed point-mutants (alanine to phenylalanine) in the predicted, tightly packed TM domain dimer interface. We hypothesized that the bulky residues should destabilize the closed conformation and eliminate the ∼3 kcal/mol energy barrier to TM domain opening and the ∼2 kcal/mol energy difference between the closed and open states, thus oversensitizing the receptor. To test this, we used all-atom molecular dynamics simulations of the isolated TM domain in explicit lipid bilayers coupled to thermodynamic potential of mean force calculations. We showed that single point mutants at the interface altered the energy landscape as predicted, but were not enough to completely eliminate the barrier to opening. However, the computational model did predict that a double mutation ati,i+4 positions at the center of the TM domain dimer eliminates the barrier and stabilizes the open conformation relative to the closed. We tested these mutants in cells with time-resolved fluorescence resonance energy transfer and death assays, and show remarkable agreement with the calculations. The single mutants had a small effect on TM domain separation and cell death, whereas the double mutant significantly increased the TM domain separation and more than doubled the sensitivity of cells to ligand stimulation.