Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region
Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region
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DOI：
10.1073/pnas.1205788109
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发表时间：
2012-10-09
影响因子：
11.1
通讯作者：
Avis, Johanna M.
中科院分区：
文献类型：
--
作者：
Stephenson, Natalie L.;Avis, Johanna M.
保守的Notch信号通路在组织的发育和自我更新中发挥着至关重要的作用。 Notch 在配体诱导的 Notch 负调控区 (NRR) 构象变化后被激活，揭示关键的蛋白水解位点 (S2) 并促进下游事件。迄今为止，这种信号激活的分子机制尚未确定。然而，强有力的间接证据支持这样一种模型：Notch 胞外域的转内吞作用与配体进入带有配体的细胞紧密相关，对 NRR 施加力以驱动所需的结构变化。在这里，我们证明对人类 Notch2 NRR 施加力确实可以暴露 S2 位点，并且最重要的是，允许金属蛋白酶 TACE（TNF-α 转换酶）进行切割。通过原子力显微镜和对人类 Notch2 NRR 的分子动力学模拟，实现了对这一过程的分子洞察。数据显示其组成的 LNR（Lin12-Notch 重复序列）和 HD（异二聚化）结构域近乎顺序地展开，其作用力与其他具有承载作用的蛋白质结构域观察到的力相似。 S2 位点的暴露是展开途径上的第一个力“屏障”，发生在任何结构域展开之前，并通过从 HD 结构域中去除 LNRA:B 接头区域来实现。金属离子增加了 Notch2 NRR 对强制展开的抵抗力，它们的去除明显有利于在较低的力下展开。结果直接证明了力介导的Notch S2位点暴露和裂解，从而牢固地确立了配体诱导Notch激活的机械转导机制的可行性。
The conserved Notch signaling pathway plays crucial roles in developing and self-renewing tissues. Notch is activated upon ligandinduced conformation change of the Notch negative regulatory region (NRR) unmasking a key proteolytic site (S2) and facilitating downstream events. Thus far, the molecular mechanism of this signal activation is not defined. However, strong indirect evidence favors a model whereby transendocytosis of the Notch extracellular domain, in tight association with ligand into the ligand-bearing cell, exerts a force on the NRR to drive the required structure change. Here, we demonstrate that force applied to the human Notch2 NRR can indeed expose the S2 site and, crucially, allow cleavage by the metalloprotease TACE (TNF-alpha-converting enzyme). Molecular insight into this process is achieved using atomic force microscopy and molecular dynamics simulations on the human Notch2 NRR. The data show near-sequential unfolding of its constituent LNR (Lin12-Notch repeat) and HD (heterodimerization) domains, at forces similar to those observed for other protein domains with a load-bearing role. Exposure of the S2 site is the first force "barrier" on the unfolding pathway, occurring prior to unfolding of any domain, and achieved via removal of the LNRA: B linker region from the HD domain. Metal ions increase the resistance of the Notch2 NRR to forced unfolding, their removal clearly facilitating unfolding at lower forces. The results provide direct demonstration of force-mediated exposure and cleavage of the Notch S2 site and thus firmly establish the feasibility of a mechanotransduction mechanism for ligand-induced Notch activation.