importantly, the simulations predict that mechanical force can further drive local unfolding of strand b5 in the HD domain in a fashion that renders the S2 site exposed for cleavage. Remarkably, the partially unfolded state with exposed S2 site appears to persist for a significant period of time and, based on analysis of the underlying folding free energy surface, it represents a weakly stable intermediate inherent to the HD domain. Physics-based atomistic simulations further suggest that the b5 strand responds dynami- cally to the disengagement of the LNR modules and, in the absence of the LNRs, it becomes decoupled from the rest of the HD domain and thus poised to sense further mechanical stress. Taken together, these results support that the HD domain itself can provide additional mechanosensing for Notch activation and that mechanical force alone might be sufficient to fully activate Notch. However, it is also possible that the elevated spontaneous structural fluctuations alone in and near the S2 region, as sug- gested by HX-MS [9] and the current simulations, could provide sufficient protease access for subsequent Notch activation. Remarkably, 40% to 50% of human T-cell acute lymphocytic leukemias harbor mutations within the NRR of Notch1 [47–49]. These are activating mutations, which increase ligand-indepen- dent Notch1 signaling via two different mechanisms [22,50]. Class II mutations include insertions of ,12 residues near the C- terminal end of the HD domain that duplicate the S2 site and directly enhance ligand-independent S2 cleavage. Class I muta- tions map to the HD domain and are typically single amino acid substitutions. Interestingly, while most of class I mutations lie in the hydrophobic core of the HD domain and cause its substantial destabilization, other class I mutations are positioned at the inter- face with the LNR domains and cause less destabilization of the HD domain [22,50]. The activating effect of the later mutations is fully consistent with the results of our simulations, which show that the disengagement of the LNR domains changes the internal dynamics of the HD domain and facilitates the access to the S2 site. Another interesting result of our simulations is the mechanistic and temporal resolution between the local unfolding of strand b5 and physical separation of HDN (plus LNRs) from HDC. The SMD simulations predict that the force required to disassociate the HD domain is not stronger than the force required for disengaging the LNR modules. Therefore, if endocytosis can mechanically unwrap the NRR fold, it ought to be able to disassociate the HD domain without the S2 cleavage. This is consistent with an important discovery by Nichols et al. [51] that endocytosis alone can physically disassociate the HD domain even without ADAM activity. Our simulations further indicate that, upon mechanical force, local unfolding of the b5 strand occurs first, and this step is then followed by separation of the HDN and HDC fragments. Therefore, if ADAM10/17 are available, the S2 cleavage most likely occurs prior to the dissociation of the HDN and HDC fragments. As the HDN and HDC fragments arise from the cleavage at the S1 site, our results provide further insight into the role of the S1 cleavage in Notch activation. The S1 cleavage is mediated by furin-like proteases in the Golgi and creates a discontinuity in Notch polypeptide chain transported to the cell surface. Some Notch1 mutants resistant to S1 cleavage were found to be defective of signaling when expressed in mammalian cells [51,52]. This led to suggestion that physical separation of the extracellular and transmembrane Notch subunits during ligand endocytosis is a prerequisite for the subsequent processing at the S2 site [51]. Other reports have demonstrated, however, that while S1 cleavage mutants show impaired intracellular trafficking, they are fully capable of signaling when present at the cell surface [9]. Thus, in the context of full-length Notch1, separation of the Mechanosensing in Notch extracellular and transmembrane domains does not seem to be required for the S2 cleavage and Notch activation. This is fully consistent with the prediction from the simulations that only local unfolding of the HD domain appears to be necessary for pro- teolytic access to the S2 site. The large size of Notch receptors, their posttranslational modifications, unusually large number of extracellular domains, and a sequence of coordinated proteolytic events add to the complexity of Notch signaling and pose many challenges in both biochemical and computational analyses of Notch activation. Some of the unresolved questions are: What is the role of multiple of EGF-like repeats? Do they function to increase pulling force exerted on the NRR during ligand endocytosis? What is the effect of receptor (and ligand) glycosylation on the mechanical properties of Notch? As the extracellular domain of Notch1 has been recently shown to form dimers [53], how does the dimerization affect mechanosensing? What is the effect of oncogenic T-ALL muta- tions on the mechanical properties and internal dynamics of Notch1? Application of computational approaches should help answering these questions and thus complement the ongoing biochemical and cell biological characterization of Notch proteins. Materials and Methods X-ray structures and domain definitions We chose to use the high-resolution X-ray structure of human Notch1 NRR (PDB: 3eto [22]) as the model of the auto-inhibited conformation. This structure was determined using a continuous construct without the S1 loop (residues 1623–1669) excised. The structure for the furin-cleaved Notch1 NRR is also available (PDB: 3i08 [9]), but with much lower resolution (3.2 A˚ vs. 2.0 A˚ for 3eto). Interestingly, the S1 loop excised in 3eto is also missing in 3io8, presumably due to the lack of a stable conformation. The structured regions of the two structures show little change except for a few residues at the S1 chain break site. Therefore, 3eto instead of 3i08 was used in the current study. The domain boundaries are: LNR-A: residues 1449–1480; LNR-B: residues 1491–1522; LNR-C: residues 1529–1562; and, HD: residues 1568–1727. The actual S1 cleavage would break the covalent linkage between residues 1622 and 1670 in the 3eto construct, and leads to two non-covalently associated subunits of the HD domain. These two subunits are designated as HDN (residues 1568–1622) and HDC (residues 1670–1727). The S2 site locates between A1721 and V1722, which is part of strand b5 of the HD domain. Topology-based coarse-grained modeling of human Notch1 NRR A ‘‘sequence-flavored’’ Go¯ -like model [42] of human Notch1 NRR was first generated based on the X-ray structure (PDB: 3eto), using the Multiscale Modeling Tools for Structural Biology (MMTSB) Go¯ -Model Builder (http://www.mmtsb.org). Each amino acid residue is represented as a single coarse-grain Ca bead connected via virtual bonds (Fig. 1c). The mass of each bead is taken to be that of the corresponding amino acid residue. As in a typical Go¯ -model, all virtual bonds and angles are derived directly from the native structure, and non-bonded Ca bead interact favorably only between pairs of residues that are in contact in the native structure. The native contacts include backbone hydrogen bonding as well as side chain interactions. However, unlike conventional Go¯-models where all native interactions contribute uniformly to the total energy, the sequence-flavored’’ Go¯ -like model incorporates the Miyazawa-Jernigan (MJ) statistical poten- tials [54] to provide residue-specific energetic biases for native interactions. In addition, the model includes knowledge-based, PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e22837