the N-terminal LNR-A detached first. This is consistent with intuitive expectations based on the examination of the NRR fold alone [21,22], as well as the proposed mechanical mechanism of Notch activation. The tertiary structures of all domains of the NRR, particularly that of the HD domain, remain largely intact throughout these initial stages of disengaging the LNR modules. Therefore, the S2 site remains inaccessible to ADAM proteases. Interestingly, the simulations predict that continuing to stretch the NRR would eventually induce local unfolding of the HD domain before dissociation of the Notch1 heterodimer. The local unfolding mainly involves the N-terminal strand b5, and the S2 site becomes fully exposed in this intermediate state. Therefore, we postulate that the putative mechanical force generated by ligand endocytosis does not only detach the protective LNR modules from the HD domain, but also further drives local unfolding of the HD domain in a functionally relevant fashion that would provide full protease access to the S2 site. We further tested this conjecture by examining the folding free energy surface of the HD domain, as well as intrinsic conformational dynamics of the HD domain with and without the LNR modules using physics-based atomistic simulations. The results support that the HD domain possesses inherent structural and dynamic characteristics that could enable it to provide additional mechanosensing necessary for Notch activation. While this work was under review, a related study was published by Tiyanont et al. on the Notch1 NRR unfolding induced by Ca2+ chelation and detected by the hydrogen-exchange mass spectros- copy (HX-MS) [41]. The results indicate that the HD domain remains stable after EDTA-induced unfolding of the LNR repeats, which is also suggested by our atomistic and coarse-grained simulations. A previous NMR study on the human Notch2 HD Mechanosensing in Notch domain alone also suggests that the whole HD domain is stably folded with and without S1 cleavage [9]. More interestingly, the HX-MS data also reveal elevated deuteration in and near the S2 region. Although significant differences in the 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. Results Characterization of the Go¯ -like model of human Notch1 NRR As detailed in the Materials and Methods, a ‘‘sequence- flavored’’ coarse-grained Go¯ -like model [42] of human Notch1 NRR was first constructed based on the X-ray structure (PDB: 3eto) [22], and then modified to include the disulfide bonds and bound calcium ions. A 1.0 ms equilibrium simulation at 300 K was performed to verify the structural stability and to characterize the dynamic properties of the NRR. 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 A˚ throughout the simulation (data not shown). The calculated root-mean-square fluctuation (RMSF) profile, shown in Fig. 2a, agrees qualitatively with the one derived from the crystal B-factors. This is expected, as the native structure is well conserved during the simulation. However, the coarse-grained simulation does appear to yield significantly higher fluctuation in loop regions as well as LNR-A and LNR-C regions. Importantly, higher (loop) flexibility does not appear to be an Figure 2. Residue RMSF profiles. a) Ca RMSF profiles calculated from the G o¯-like and GBSW simulations, in comparison with the one derived from the X-ray B-factors. The final 80% of the trajectories (1.0 ms for G o¯-like and 20 ns for GBSW) were used in the RMSF calculations. All snapshots were globally aligned to minimize the RMSD with respect to the native structure. The crystal B factors were converted into RMSF values using the relationship B-factor = 8p2(RMSF)2/3. b) Ca RMSF profiles for the HD domain only, calculated from the last 16 ns of the 20 ns GBSW simulations of the full NRR and HD domain only. Note that, to better reflect the fluctuations within the HD domain with and without the LNR modules, the RMSF profile for the full NRR simulation was recalculated by aligning all snapshots to minimize the RMSD of the HD domain (instead of the full NRR in Panel a). Residue ID 1–174 and 175–232 shown correspond to residues 1449–1622 and 1670–1727 in human Notch1 numbering, respectively. doi:10.1371/journal.pone.0022837.g002 PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22837