weakening of strand b5 once the LNR modules, particularly, the LNR-AB linker, are disengaged from the HD domain. We further examined the folding free energy surfaces of the HD domain using the Go¯ -like model. A 1.0-ms replica exchange MD (REX-MD) simulation was preformed to extensively sample the accessible conformational space and allow converged free energy surfaces to be computed using the weighted histogram analysis method (WHAM) [43–45]. Fig. 6 plots the free energy surface near the melting temperature (Tm,350 K), as a function of the fraction of all native contacts (Q) and that of native contacts formed by strand b5 (Qb5). It reveals that the HD domain naturally unfolds through a weakly stable intermediate state (marked as I in Fig. 6), where strand b5 is largely unfolded (Qb5,0.1) while the rest of the protein is still mostly folded. Importantly, transition between the intermediate and unfolded states involves a free energy barrier that is substantially higher than that between the folded and intermediate states. This is consistent the observation that significantly higher extension force is required to fully unfold (and disassociate) the HD domain. Furthermore, the intermediate state identified in the equilibrium calculation is similar to the one observed in force-induced unfolding trajectories (e.g., see Fig. 3 and Fig. 4). Therefore, external mechanical force induces a natural unfolding pathway of the HD domain that provides transient proteolytic access to the S2 site. In other words, the HD domain possesses inherent folding characteristics that enable it to respond to mechanical stress in a functionally relevant way. Intrinsic conformational dynamics of the HD domain Two 20 ns atomistic simulations were performed to further investigate the overall motions and fluctuations of the full NRR and the HD domain alone, respectively. The purpose is to explore potential links between dynamics and mechanosensing. An optimized GBSW implicit solvent force field [46] was used for computational efficiency. As summarized in Fig. 7, both the NRR and the HD domain alone were stable throughout the 20 ns simulation timescale. The backbone RMSD from the X-ray structure of the NRR quickly increased and stabilized around Figure 6. Folding free energy surface of the HD domain. The surface is shown as a function of the total native fraction (Q) and fraction of native contacts formed by strand b5 (Qb5) near the melting temperature. The free energy surface was calculated from a 1-ms REX- MD simulation using the G o¯-like model. Contours are drawn at every kT. Approximate locations of the unfolded (U), intermediate (I), and folded (F) states are marked. doi:10.1371/journal.pone.0022837.g006 Mechanosensing in Notch 3.5 A˚. The moderate RMSD value is mainly due to fluctuations in loops and tertiary packing between the LNR repeats and the HD domain. The backbone RMSD of the HD domain itself from the X-ray structure is much smaller, ,2.5 A˚, with or without the LNR modules (green and blue traces in Fig. 7a). All secondary structure elements in the HD domain were also well maintained during both simulations (Fig. 7b). Average structures computed from the last 16 ns of the 20 ns trajectories, shown in Fig. 7c and 7d, illustrate that the HD domain itself is stable. Absence of the LNR modules only leads to small local conformational changes at the interface, with one of the most notable changes being a slight distortion of helix a3 (marked by arrows in Fig. 7d). The helix a3 distortion is apparently a direct consequence of the hydrophobic void created by removal of the LNR-AB linker (Fig. 5). While these atomic simulations with limited length are not sufficient to establish the protein stabilities, they are consistent with earlier notions that the HD domain itself appears to be stable and that (mechanically) lifting of the LNR modules is not likely to provide sufficient proteolytic access to the S2 site. Furthermore, a separate simu- lation shows that a chain break in the S1 loop does not appear to destabilize the isolated HD domain (see Fig. S1). The prediction of a stable isolated human Notch1 HD domain has since been confirmed by the recent HX-MS study [41] and is also consistent with previous NMR studies of the human Notoch2 HD domain [9]. Interestingly, in contrast to the apparent structural stability and insensitivity to removal of the LNR modules, internal dy- namics of the HD domain appears to respond in a sensitive man- ner to the absence of the LNR modules. Residue RMSF profiles, shown in Fig. 2b, reveal that there is a significant increase in fluctuations in the exposed HD domain, especially at the termini and interfacial loops. Changes in protein dynamics are even more pronounced when one examines correlated motions by calculating the covariance between fluctuations of two residues. Fig. 8 com- pares the covariance matrices for the HD domain in the presence (Fig. 8a) and absence of the LNR modules. The covariance matrix for the full NRR is shown in Fig. S2. Clearly, there is not only a significant increase in the overall degree of dynamic coupling in the free HD domain, but the pattern of correlated motions also changes substantially. However, the C-terminal strand b5 (mapped to residue ID 226–232) becomes only weakly coupled the rest of HD domain in the absence of the LNR modules (e.g., to strands b2, b3 and b4; marked by dashed boxes in Fig. 8), while the preceding loop (mapped to residue ID 214–224) becomes more tightly coupled to adjacent structural elements and subsequently to the rest of the protein. Therefore, the HD domain responds dynamically to disengagement of the LNR modules in a fashion that it becomes poised to sense further mechanical stress via activation of an inherent unfolding pathway, namely, local unfolding of the C- terminal strand b5 to expose the S2 site prior to the disassociation of the HD domain. Discussion The key event in the activation of Notch signaling is ADAM- mediated cleavage of the receptor at the S2 site. As the S2 site is buried within the HD domain and is not available for cleavage, an outstanding question remains: How does ligand engagement overcome auto-inhibition of the ‘‘off’’ state? Two unique features of the Notch signaling system are that the receptor and the ligand are located on two opposing cells and that receptor-ligand inter- action is followed by ligand endocytosis into the ligand-expressing cell. This invites a question about force-induced conformational changes in the receptor. The mechanostransduction model of PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22837