Mechanotransduction of the Notch Signalling Pathway via the Negative Regulatory Region

The Notch receptor is part of a highly conserved omnipresent developmental pathway that has crucial roles in developing and self-renewing tissues. During activation of the signalling pathway Notch binds to its ligand, presented on a neighbouring cell. It is thought that this results in a conformational change within the Negative Regulatory Region (NRR) unmasking a key proteolytic site (S2) and allows for metalloprotease cleavage. This facilitates further cleavage by gamma-secretase, initiating downstream events. Thus far, the molecular mechanism by which the S2 site is revealed has not been defined, though indirect evidence favours a model whereby transendocytosis of the Notch extracellular domain into the ligand bearing cell results in mechanical unfolding of the NRR. Research presented here suggests the NRR of human Notch2 (hN2) unfolds within a mechanosensing force range. Furthermore, through the application of a force (200 pN) the hN2-NRR was shown to unfold sufficiently to expose the S2 site allowing cleavage by metalloproteases. Molecular dynamics (MD) simulations offer insight into the unfolding process of the hN2-NRR, revealing near-sequential unfolding of its constituent LNR and HD domains. Removing the linker region between LNR? s A and B appears to be the first force ? barrier? in the unfolding pathway, producing the largest increase in solvent accessibility at the S2 cleavage site. Through docking simulations, this unfolding event was shown to expose the S2 cleavage site sufficiently to allow access to the metalloprotease TACE. Removing coordinated metal ions from the hN2-NRR structure resulted in a dramatic decrease in the forces required for unfolding during AFM experiments, highlighting their role in increasing the resistance of the hN2-NRR to forced unfolding. Removal of disulphide bonds within the structure resulted in a loss of detectable LNR unfolding, highlighting their role in LNR stabilisation.Six HD destabilising mutants, characterised through their role in the hN1 disease, T-cell Acute Lymphoblastic Leukemia, showed three key changes to the unfolding pathway of the hN2-NRR. Firstly, mutants A1647P, L1573P and V1623D showed a dramatic decrease in force required for unfolding in AFM experiments. MD simulations highlighted a lack of force required for the unfolding the LNRA:B linker previously characterised as the key event in removing NRR autoinhibition. Secondly, all the mutants studied here showed changes to the stability of the alpha3-helix (within the HD domain) resulting in transient shifts or bending during unfolding of the LNRA:B linker and the LNRB. Finally, changes were observed within the LNRC of A1647P and L1566P. Within these mutants the LNRC was observed to be unfolding, an event not present during wild-type unfolding. Within mutant L1566P this is thought to be due to the disruption of the conserved salt-bridge occurring between Arg1567 (HD domain) and Asp1506 (LNRC). Within mutant A1647P this is likely due to widespread domain destabilisation. Overall, research presented here has provided the first direct evidence that the NRR is mechanosensing and that mechanical force can allow for cleavage at the S2 site. Further characterisation has been performed to analyse the unfolding pathway through ion chelation, disulphide oxidation and mutagenesis studies.