Force-Induced Unfolding Simulations of the Human Notch1 Negative Regulatory Region: Possible Roles of the Heterodimerization Domain in Mechanosensing Jianhan Chen*, Anna Zolkiewska* Department of Biochemistry, Kansas State University, Manhattan, Kansas, United States of America Abstract Notch receptors are core components of the Notch signaling pathway and play a central role in cell fate decisions during development as well as tissue homeostasis. Upon ligand binding, Notch is sequentially cleaved at the S2 site by an ADAM protease and at the S3 site by the c-secretase complex. Recent X-ray structures of the negative regulatory region (NRR) of the Notch receptor reveal an auto-inhibited fold where three protective Lin12/Notch repeats (LNR) of the NRR shield the S2 cleavage site housed in the heterodimerization (HD) domain. One of the models explaining how ligand binding drives the NRR conformation from a protease-resistant state to a protease-sensitive one invokes a mechanical force exerted on the NRR upon ligand endocytosis. Here, we combined physics-based atomistic simulations and topology-based coarse-grained modeling to investigate the intrinsic and force-induced folding and unfolding mechanisms of the human Notch1 NRR. The simulations support that external force applied to the termini of the NRR disengages the LNR modules from the heterodimerization (HD) domain in a well-defined, largely sequential manner. Importantly, the mechanical force can further drive local unfolding of the HD domain in a functionally relevant fashion that would provide full proteolytic access to the S2 site prior to heterodimer disassociation. We further analyzed local structural features, intrinsic folding free energy surfaces, and correlated motions of the HD domain. The results are consistent with a model in which the HD domain possesses inherent mechanosensing characteristics that could be utilized during Notch activation. This potential role of the HD domain in ligand-dependent Notch activation may have implications for understanding normal and aberrant Notch signaling. Citation: Chen J, Zolkiewska A (2011) Force-Induced Unfolding Simulations of the Human Notch1 Negative Regulatory Region: Possible Roles of the Heterodimerization Domain in Mechanosensing. PLoS ONE 6(7): e22837. doi:10.1371/journal.pone.0022837 Editor: Markus J. Buehler, Massachusetts Institute of Technology, United States of America Received February 14, 2011; Accepted June 30, 2011; Published July 28, 2011 Copyright: 2011 Chen, Zolkiewska. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NSF through a CAREER Award (MCB 0952514) to JC and by the National Institutes of Health through an R15 Award (CA151065) to AZ. This work is contribution 11-225-J from the Kansas Agricultural Experiment Station. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jianhanc@ksu.edu (JC); zolkiea@ksu.edu (AZ) Introduction Notch signaling is a highly conserved inter-cellular communi- cation pathway that plays critical roles in embryonic development and in tissue homeostasis [1–3]. Abnormal Notch signaling is frequently associated with a number of human diseases, including T-cell acute lymphocytic leukemia (T-ALL) and solid tumors [4,5]. At the heart of Notch signaling are the Notch receptors, a family of highly modular, single-pass transmembrane proteins [6,7]. There are four Notch receptors in mammals, designated as Notch1–4. All Notch receptors have a similar modular architecture, as illustrated in Fig. 1a for human Notch1. During maturation, the Notch receptor is cleaved at the S1 site by a furin-like protease, which yields a heterodimer with non-covalently associated extracellular and transmembrane subunits [8,9]. Canonical Notch activation is initiated by binding of a transmembrane ligand of the Delta/ Serrate/LAG-2 (DSL) family on the signal-sending cell to a Notch receptor on the surface of the signal receiving cell [2,6,7]. Through mechanisms yet to be precisely determined, ligand engagement triggers regulated intra-membrane proteolysis where the Notch receptor is first cleaved at a juxtamembrane extracellular site S2 by ADAM10 or ADAM17 metalloproteases [10–14]. After the S2 cleavage, the Notch receptor is further cleaved at an intra- membrane S3 site by the c-secretase complex [15–17]. The S3 cleavage releases the intracellular portion of Notch (ICN) and allows it to translocate to the nucleus and participate in tran- scription regulation in a context and gene dose dependent manner [18,19]. Important progresses have been made towards understanding a structural basis of Notch signaling and regulation [7,20]. One of the key advances is the recent determination of high-resolution X- ray structures of the negative regulatory regions (NRR) of human Notch1 and Notch2 [21,22]. The NRR consists of three Lin12/ Notch repeats (LNR) and the heterodimerization (HD) domain. The later contains both the S1 and S2 sites near the middle and the C-terminus, respectively (Fig. 1). The X-ray structure (Fig. 1b) reveals that the NRR maintains an auto-inhibited, compact fold where the LNR modules wrap around the HD domain and shield the S2 site from metalloprotease access. Clearly, large-scale conformational movements involving a displacement of LNR-A, the LNR-AB linker, and LNR-B from the HD domain are necessary for an ADAM protease to gain access to the S2 site. Several models have been proposed to explain how ligand binding might facilitate large conformational changes within the NRR, PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22837