Mechanosensing in Notch Figure 1. Overview of the human Notch1 domain organization and structure. a) The domain organization of human Notch1 receptor. The N-terminus includes 36 EGF-like repeats, followed by the NRR, a short transmembrane (TM) segment, and the ICN. Approximate locations of the proteolytic cleavage sites S1, S2 and S3 are marked. b) The X-ray structure of human Notch1 NRR (PDB:3eto [22]). The N-terminal LNR domains A–C are colored in blue, green and red, respectively. All disulfide linkages and three bound calcium ions are shown. The HD domain is colored based on the local secondary structure, with the S2 cleavage site and adjacent secondary structural elements (b1, b5 and a3) marked. c) A Ca-only model of human Notch1 NRR derived from the X-ray structure. doi:10.1371/journal.pone.0022837.g001 including the allosteric model and the mechanotransduction model [6,7]. According to the allosteric model, ligand binding leads to major rearrangements of the LNR modules with respect to the HD domain. An allosteric mechanism is more likely to occur in receptors with relatively short extracellular domains, such as Notch receptors in C.elegans. In mammals and flies, however, where the extracellular domains of Notch receptors contain a large number (29–36) of epidermal growth factor (EGF) repeats and where the ligand binding site (EGF repeats 11–12) is positioned relatively far from the NRR, such long-range allosteric changes seem less likely. The mechanotransduction model exploits the fact that a receptor- bound DSL ligand undergoes endocytosis into the signal-sending cell and pulls on the Notch receptor present in the signal-receiving cell. The Notch ectodomain is effectively trans-endocytosed into the ligand-expressing cell, while its transmembrane and intracellular domains remain associated with the receptor-expressing cell [23,24]. Thus, the mechanotransduction model proposes that ligand endocytosis generates a mechanical strain on the NRR region and leads to ‘‘peeling off’’ the protective LNR modules from the HD domain [21,22]. This model is fully consistent with previous studies demonstrating that ligand endocytosis is indeed necessary for Notch activation [25,26] and that free ligands are generally not capable of activating Notch receptors [27,28]. It has to be noted, however, that the requirement for ligand endocytosis has been also interpreted as a need for additional ligand processing within the endocytic com- partment in order to potentiate the ligand signaling activity [29,30], which is not related to receptor pulling. Regardless of the mechanism involved in the rearrangement of the LNR modules during ligand-induced Notch activation, the location of the S2 site within the folded HD domain poses additional barrier for the access by ADAM metalloproteases. The S2 site is buried in a hydrophobic groove (in the middle of strand b5 beneath helix a3; see Fig. 1b). While the specific structural signatures for recognition by ADAM metalloproteases are yet to be established [31], the active sites of these proteases lie in deep clefts [32]. Clearly, unwrapping of the LNR modules alone is not sufficient to provide proteolytic access to the scissile bond at the S2 site. It has been postulated that either local or global unfolding of the HD domain must follow the displacement of the LNR modules to expose the S2 site [22]. However, the driving force and possible mechanisms for either local or global unfolding of the HD domain are not known. In this work, we exploited topology-based coarse-grained and physics-based atomistic molecular dynamics (MD) simulations to study the conformational dynamics of the Notch NRR at equili- brium and under mechanical stress. Such computer simulations can provide a unique opportunity to directly observe protein un- folding pathways and potential short-lived intermediate states that are not accessible experimentally [33–35]. In particular, steered molecular dynamics (SMD) using simple topology-based coarse- grained models has been shown to be capable of reliable de- scription of mechanically induced unfolding of proteins [36]. Topology-based modeling is built on the conceptual framework of minimally frustrated energy landscapes [37,38]. Minimal frustration argues that natural proteins achieve efficient and robust folding by possessing largely smooth, funneled underlying free energy landscapes. There is a strong correlation between the fraction of native contacts formed and free energy. That is, native interactions largely shape the protein energy landscape. Therefore, for a given protein with known structure, one might construct simple effective energy functions by taking only native interactions into account. These topology-based models are often referred to as Go¯ or Go¯ -like models. Impressive correspondence between experi- ment and theory for folding mechanisms has been demonstrated for many proteins [39,40], substantiating the notion that protein topology dictates gross aspects of the folding mechanism (e.g., order of events in folding). We first investigated the unfolding pathway of human Notch1 NRR under mechanical force using a Go¯ -like model derived from the X-ray structure. SMD simulations show that pulling on the N- and C-termini of human Notch1 NRR lifts the LNR modules from the HD domain in a well defined, largely sequential fashion with PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22837