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
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