Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region

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.