Mechanosensing in Notch Figure 4. Representative snapshots of the human Notch1 NRR along the force-induced unfolding pathway during one of the SMD pulling simulations. The LNR-A, LNR-B and LNR-C modules are colored in blue, green and red, respectively, and the HDN and HDC subunits of the HD domain in cyan and purple, respectively. The N- and C-terminal Ca beads where the external force is exerted are shown in black spheres. The S2 site is marked using purple spheres. Approximate end-to-end distances for the snapshots are also listed. Note that the magnified view of the 166 A˚ snapshot is slightly rotated to better show the exposure of the S2 site. doi:10.1371/journal.pone.0022837.g004 modules (e.g., see Fig. 4 for a representative snapshot at 75 A˚ end- to-end displacement). Therefore, unwrapping the LNR modules alone is not likely to provide sufficient proteolytic access to the S2 site, as expected [21,22]. Further pulling continues to stretch the entropic loops connecting these sub-domains. Interestingly, the next key unfolding event is predicted to be local unfolding of the C-terminal strand b5, which occurs in a cooperative fashion when the extension force approaches ,20 pN (Peak 3 in Fig. 3a). As illustrated by a representative snapshot shown in Fig. 4 (with ,166 A˚ end-to-end distance), local unfolding of b5 fully exposes the S2 site for proteolytic access. This suggests that mechanical force generated by ligand endocytosis is capable of fully relieving the auto-inhibition for Notch activation. Eventual disassociation of the Notch heterodimer is predicted to occur in two stages, mainly involves unfolding of the secondary structure elements a3 and b4, respectively. The two-stage dissociation leads to two broad force peaks (Peaks 4 and 5 in Fig. 3a). It is noteworthy that heterodimer dissociation requires an extension force of ,40 pN, considerably higher than what is required to locally unfold the b5 strand. This suggests that the intermediate state with exposed S2 site can persist for a significant period of time before global unfolding and heterodimer disassociation. An important implication is that the partially unfolded state might be required for specific recognition by an ADAM metalloprotease. Intrinsic folding characteristics of the HD domain The SMD pulling simulations reveal that, in addition to the auto-inhibited NRR fold, the HD domain also plays a central role in mechanosensing and Notch activation. To better understand the intrinsic folding characteristics of the HD domain, we first analyzed the structural basis of the observed mechanical weakness of the C-terminal strand b5. As illustrated in Fig. 5, strand b5 only contains two small hydrophobic residues (A1721 and V1722), while all other strands in the HD domain contain several and larger hydrophobic residues. For example, the adjacent strand b1 contains six consecutive leucine and valine residues from L1575 through L1580. Furthermore, strand b5 is loosely packed with a face of the C-terminal helix a3 that is lined with alanine and glycine residues (A1697, A1702, G1795, A1708) and thus completely lacks any large (hydrophobic) side chains that could provide stabilizing contacts with strand b5. Instead, this local void in the hydrophobic core is filled by L1482 and F1484 from the LNR-AB linker (see Fig. 5). In addition, the LNR-AB further stabilizes the NRR fold through a hydrogen bonding interaction from N1483 to E1720 on the loop that precedes the HD strand b5. These structural features fully explain substantial local mechanical Figure 5. Local structural features near the HD domain strand b5. All heavy atoms of hydrophobic side chains of the HD domain are shown in gray sticks. The LNR modules are drawn in semi-transparent colors to illustrate a lack of stabilizing contacts of strand b5 in the absence of the LNR-AB liner. The key residues discussed in the main text are labeled. doi:10.1371/journal.pone.0022837.g005 PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22837