Force-Induced Unfolding Simulations of the Human Notch1 Negative Regulatory Region: Possible Roles of the Heterodimerization Domain in Mechanosensing

Notch receptors are core components of the Notch signaling pathway and play a central role in cell fate decisions during development as well as tissue homeostasis. Upon ligand binding, Notch is sequentially cleaved at the S2 site by an ADAM protease and at the S3 site by the γ-secretase complex. Recent X-ray structures of the negative regulatory region (NRR) of the Notch receptor reveal an auto-inhibited fold where three protective Lin12/Notch repeats (LNR) of the NRR shield the S2 cleavage site housed in the heterodimerization (HD) domain. One of the models explaining how ligand binding drives the NRR conformation from a protease-resistant state to a protease-sensitive one invokes a mechanical force exerted on the NRR upon ligand endocytosis. Here, we combined physics-based atomistic simulations and topology-based coarse-grained modeling to investigate the intrinsic and force-induced folding and unfolding mechanisms of the human Notch1 NRR. The simulations support that external force applied to the termini of the NRR disengages the LNR modules from the heterodimerization (HD) domain in a well-defined, largely sequential manner. Importantly, the mechanical force can further drive local unfolding of the HD domain in a functionally relevant fashion that would provide full proteolytic access to the S2 site prior to heterodimer disassociation. We further analyzed local structural features, intrinsic folding free energy surfaces, and correlated motions of the HD domain. The results are consistent with a model in which the HD domain possesses inherent mechanosensing characteristics that could be utilized during Notch activation. This potential role of the HD domain in ligand-dependent Notch activation may have implications for understanding normal and aberrant Notch signaling.

Notch受体（Notch receptors）是Notch信号通路（Notch signaling pathway）的核心组成部分，在发育过程中的细胞命运决定以及组织稳态中发挥核心作用。配体结合后，Notch会依次经ADAM蛋白酶在S2位点、γ-分泌酶复合物（γ-secretase complex）在S3位点被切割。近期解析的Notch受体负调控区域（negative regulatory region, NRR）的X射线晶体结构显示，其呈现自抑制构象：NRR中三个具有保护作用的Lin12/Notch重复序列（Lin12/Notch repeats, LNR）会遮蔽位于异二聚化结构域（heterodimerization domain, HD）中的S2切割位点。现有一种解释配体结合如何使NRR构象从蛋白酶抗性状态转变为蛋白酶敏感状态的模型，提出了配体内吞时会对NRR施加机械力的机制。本研究结合基于物理的全原子模拟与基于拓扑的粗粒化建模方法，探究了人源Notch1 NRR的固有折叠、解折叠以及力诱导折叠/解折叠机制。模拟结果表明，施加于NRR末端的外力会以明确且高度有序的分步方式，使LNR模块与HD结构域分离。值得注意的是，机械力还能以符合生理功能的方式诱导HD结构域发生局部解折叠，从而在异二聚体解离前，让S2位点完全获得蛋白酶切割的可及性。本研究还进一步分析了HD结构域的局部结构特征、固有折叠自由能面以及关联运动模式。研究结果与下述模型一致：HD结构域具有固有的机械感知特性，该特性可在Notch激活过程中被利用。HD结构域在配体依赖型Notch激活中的这一潜在作用，或可为理解正常及异常的Notch信号通路提供新的理论参考。