Abstract
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.
Highlights
Notch signaling is a highly conserved inter-cellular communication pathway that plays critical roles in embryonic development and in tissue homeostasis [1,2,3]
Significant differences in the negative regulatory region (NRR) unfolding pathway may exist when unfolding is induced by a chemical agonist or by a mechanical force, the results reported by Tiyanont et al are remarkably consistent with one of the key predictions of our simulations, that the HD domain has the inherent ability to partially unfold at the C-terminal strand
The results show that the protein is stable in the Go-like coarse-grained effective potential as designed, with the root-mean-square deviation (RMSD) from the native structure fluctuating around 3 Athroughout the simulation
Summary
Notch signaling is a highly conserved inter-cellular communication pathway that plays critical roles in embryonic development and in tissue homeostasis [1,2,3]. At the heart of Notch signaling are the Notch receptors, a family of highly modular, single-pass transmembrane proteins [6,7]. The Notch receptor is cleaved at the S1 site by a furin-like protease, which yields a heterodimer with non-covalently associated extracellular and transmembrane subunits [8,9]. Through mechanisms yet to be precisely determined, ligand engagement triggers regulated intra-membrane proteolysis where the Notch receptor is first cleaved at a juxtamembrane extracellular site S2 by ADAM10 or ADAM17 metalloproteases [10,11,12,13,14]. After the S2 cleavage, the Notch receptor is further cleaved at an intramembrane S3 site by the c-secretase complex [15,16,17]. The S3 cleavage releases the intracellular portion of Notch (ICN) and allows it to translocate to the nucleus and participate in transcription regulation in a context and gene dose dependent manner [18,19]
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