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 receptor）是Notch信号通路的核心组成元件，在发育进程中的细胞命运决定与组织稳态维持中发挥核心调控作用。当与配体结合后，Notch会先后被ADAM蛋白酶在S2位点、γ-分泌酶复合物在S3位点依次完成切割。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，其呈现自抑制折叠构象：NRR所包含的3个保护性Lin12/Notch重复序列（Lin12/Notch repeats, LNR）会遮蔽位于异二聚体化（heterodimerization, HD）结构域内的S2切割位点。现有用于阐释配体结合如何驱动NRR构象从蛋白酶抗性状态转变为蛋白酶敏感状态的模型中，有一类提出了配体内吞时对NRR施加机械力的作用机制。本研究结合基于物理原理的全原子模拟与基于拓扑结构的粗粒度建模，探究了人源Notch1 NRR的固有折叠、解折叠机制以及力诱导的构象变化过程。模拟结果显示，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结构域发生解离。值得注意的是，机械力还能以符合生理功能的方式驱动HD结构域发生局部解折叠，从而在异二聚体解离前完全暴露S2切割位点以供蛋白酶识别切割。本研究进一步分析了HD结构域的局部结构特征、固有折叠自由能面以及协同运动特性。所得结果与"HD结构域具备固有机械传感特性，可在Notch激活过程中被利用"的模型相符。HD结构域在配体依赖型Notch激活中的这一潜在作用，或可为理解正常与异常的Notch信号通路调控机制提供新的理论参考。