Direct observation of proteolytic cleavage at PNAS PLUS the S2 site upon forced unfolding of the Notch negative regulatory region

Natalie L. Stephenson and Johanna M. Avis1

Faculty of Life Sciences, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom

Edited by Iva Greenwald, Columbia University, New York, NY, and approved September 5, 2012 (received for review April 7, 2012)

The conserved Notch signaling pathway plays crucial roles in devel- brane region, cleaving and consequently releasing the Notch oping and self-renewing tissues. Notch is activated upon ligand- intracellular domain, which translocates to the nucleus to upre- induced conformation change of the Notch negative regulatory gulate its target genes. Insight into the mechanism by which region (NRR) unmasking a key proteolytic site (S2) and facilitating ligand binding results in a structure change in the Notch NRR, downstream events. Thus far, the molecular mechanism of this sufficient to facilitate S2 cleavage, is key to understanding the signal activation is not defined. However, strong indirect evidence activation process. favors a model whereby transendocytosis of the Notch extracellu- Crystal structures of the NRR region of the human Notch1 (17) lar domain, in tight association with ligand into the ligand-bearing and Notch2 (12) receptors show burial of the S2 site via wrapping cell, exerts a force on the NRR to drive the required structure of the three LNR (Lin12-Notch repeat) modules (A–) around change. Here, we demonstrate that force applied to the human the HD (heterodimerization) domain, with the connecting linker Notch2 NRR can indeed expose the S2 site and, crucially, allow clea- between LNRA and B (A∶B) forming a plug of inhibition over vage by the metalloprotease TACE (TNF-alpha-converting enzyme). the S2 cleavage site in the HD domain. Each LNR domain is Molecular insight into this process is achieved using atomic force stabilized by three disulfide bonds and a coordinated calcium ion. microscopy and simulations on the human In the human Notch2 structure, a zinc ion is also present close to Notch2 NRR. The data show near-sequential unfolding of its con- the linker of LNRB and C (B∶C) (Fig. 1). Because the key region BIOPHYSICS AND

stituent LNR (Lin12-Notch repeat) and HD (heterodimerization) for ligand binding (EGF-like repeats 11–13) (18, 19) is distal to COMPUTATIONAL BIOLOGY domains, at forces similar to those observed for other the S2 cleavage site by a further approximately 20 EGF-like re- domains with a load-bearing role. Exposure of the S2 site is the first peats in human Notch1–4, and significant parts of the EGF repeat force “barrier” on the unfolding pathway, occurring prior to un- region will be in an extended rod-like structure (20), an allosteric folding of any domain, and achieved via removal of the LNRA∶B mechanism for conformational change at S2 would seem unlikely linker region from the HD domain. Metal ions increase the resis- and, indeed, has little experimental support. Conversely, consid- tance of the Notch2 NRR to forced unfolding, their removal clearly erable evidence now exists that Notch relies on endocytosis of facilitating unfolding at lower forces. The results provide direct its ligand for signal activation in the receiving cell. A number of demonstration of force-mediated exposure and cleavage of the studies in vertebrates and flies have proposed that this ligand en- Notch S2 site and thus firmly establish the feasibility of a mechan- docytosis provides a mechanical force to unravel at least part of otransduction mechanism for ligand-induced Notch activation. the Notch NRR structure. Firstly, the forces of adhesion and dis- sociation measured between Notch-bearing and ligand-bearing mechanical force ∣ signaling receptor cells indicate an extremely tight Notch-ligand association that is proportional to signaling rates (21). Indeed, somewhat crucially, he Notch signaling pathway (1, 2), though simple and direct transendocytosis of the Notch extracellular region with ligand into Tcompared with many signaling pathways, has a diverse and the ligand-presenting cell has been observed (22, 23). Further in- crucial role in both developing tissues and adult tissues that un- direct evidence for force-mediated Notch activation comes from dergo self-renewal. The pathway functions by enabling cell–cell observations that Notch signaling is enhanced upon immobiliza- communication that, depending on signal context and dose, tion of soluble forms of ligand and that, if not immobilized, such – directs the cells to a preferential cell fate. Through this system, soluble ligands actually inhibit Notch signaling (24 26). the pathway regulates numerous cellular decisions including cell The role of mechanical force in cellular signaling mechanisms cycle progression, metabolism, and cell differentiation (3–5). is an emerging and rapidly expanding area (27). The initial sen- sing of a mechanical stimulus (28–30) occurs at a cell surface, and With such a diverse and crucial role in development and self-re- “ ” newal, it is unsurprising that mutations and errors within this sig- among identified sensors are cell adhesion molecules, integrin – receptors, ion-sensitive channels, protein kinases, and G naling pathway give rise to several diseases and cancers (6 10). – In humans, there are four Notch receptors, Notch1–4, which (31 34). Although there is increasing evidence for mechanically are very similar in domain architecture and all require proteolytic regulated signaling pathways, including the Notch pathway stu- processing for signal activation and transmission via the canonical died here, direct evidence that provides insight into the mechan- pathway. Prior to signal activation, a Notch receptor is expressed osensors and signal transduction events at a detailed molecular on the cell surface as a furin-cleaved heterodimer (furin cleaves at level is lacking. This lack of molecular detail is in part due to the site S1). A second proteolytic cleavage site, S2, is buried within challenges of applying force techniques on biological systems, the fold of the extracellular Notch negative regulatory region (NRR; Fig. 1) (11, 12), where it is protected from cleavage by Author contributions: N.L.S. and J.M.A. designed research; N.L.S. performed research; certain proteases of the ADAM (a disintegrin and metallopro- N.L.S. and J.M.A. analyzed data; and N.L.S. and J.M.A. wrote the paper. tease) family (13, 14). The binding of transmembrane ligand The authors declare no conflict of interest. (Delta/Serrate/Lag-2) (15, 16), presented on a neighboring cell, This article is a PNAS Direct Submission. to certain Notch epidermal growth factor (EGF)-like repeats, 1To whom correspondence should be addressed. E-mail: [email protected]. in some way triggers cleavage at S2. Once cleaved at this site, This article contains supporting information online at www.pnas.org/lookup/suppl/ γ-secretase can access a third site (S3) in the Notch transmem- doi:10.1073/pnas.1205788109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1205788109 PNAS Early Edition ∣ 1of9 Downloaded by guest on September 29, 2021 molecule is extended. The hN2-NRR protein construct contained an amino-terminal poly-lysine tag to optimize directed covalent attachment of this protein terminus to an N-hydroxysuccinimide (NHS)-functionalized gold surface. A carboxy-terminal hexa- histidine tag was used to direct attachment of the AFM tip (func- tionalized with Ni2þ-NTA) to the C terminus of the protein. Although a protein construct comprising NRR polymeric repeats would simplify data interpretation (35, 36), a single NRR contains 10 disulphide bonds, presenting a formidable challenge (which we could not overcome) to obtaining correctly folded polymeric pro- duct by either tandem cloning or chemical ligation routes. Hence, a single recombinant hN2-NRR stabilized by disulfide bonds and coordinating metal ions in the correctly folded conformation, as assessed by CD and two-dimensional 1H NOESY NMR, was attached between AFM cantilever tip and slide and subjected to a pulling force in the AFM. Notably, the hN2-NRR protein does in itself contain multiple domains. Representative saw-tooth force- extension curves observed upon stretching hN2-NRR single mole- cules by AFM (Fig. 2A) show features consistent with unfolding of the multiple domains within this protein fragment. In force-exten- sion curves, at least two smaller extensions, to approximately 7 nm,

Fig. 1. The Notch receptor and the NRR of hN2. (A) The domain structure of the Notch receptor contains an extracellular region, consisting of the NRR and the EGF-like repeats (green; repeats required for ligand interactions are striped). The NRR comprises three LNR modules (light pink to dark pink) and the furin-cleaved HD domain (HD-N, dark blue; HD-C, light blue). Approxi- mate positions of S2 and S3 cleavage sites, which release the intracellular domain, are shown. (B) X-ray structure of hN2-NRR in its autoinhibited conformation. The LNR and HD domains are colored as in A, each LNR con- taining a coordinated calcium ion (green) and three disulfide bonds (red lines). The zinc ion, coordinated by residues of the B∶C linker and HD domain, is shown in gray. The S2 cleavage site is highlighted by a black arrow. The furin cleavage loop (S1) is indicated by a red arrow. PDB ID code 2OO4 (12). Figure created using PyMol (version 1.3).

which will require staged approaches and combined technologies. This work represents experimental application of force on a Notch molecule, with observation of effect, aimed at directly prob- ing the feasibility of a force-induced protein unfolding mechanism for exposure of the S2 site within the NRR. We use recombinant NRR from human Notch2 (hN2), for which the crystal structure first revealed an autoinhibited conformation (12). Using atomic force microscopy (AFM), we monitor unfolding of the constituent LNR and HD domains of the hN2-NRR, validating these experi- mental observations via steered molecular dynamics (MD) simu- lations, which provide further molecular insight into structural changes as the NRR unfolds. The combination of techniques yields the greatest molecular insight thus far into the response of the Notch2 NRR to an applied force, such as it presumably experi- ences upon transendocytosis. Importantly, we correlate NRR mechanical unfolding to S2 site accessibility by the metallopro- tease TACE (TNF-alpha-converting enzyme). Direct observation Fig. 2. AFM unfolding of hN2-NRR region. (A) Raw data from AFM experi- of cleavage at S2 upon mechanical stretching of Notch is observed. ments with NRR showing distinct unfolding features corresponding to the LNR (Left) and HD domains (Right). Worm-like chain (WLC) analysis shown on Results unfolding curves (black line). Mean coefficient of determination for WLC analysis of data ¼ 0.98. The first peak was shown (via controls) to correspond Characterization of the Mechanical Properties of hN2-NRR by AFM. to salt contacts between the tip and slide, and is therefore not analyzed by Mechanical unfolding of the Notch NRR has been hypothesized the WLC. Frequency of extension (B) and force (C) observed during all unfold- to expose the S2 cleavage site, allowing metalloprotease access and ing events (LNR and HD) in the presence (striped; n ¼ 1;075) and absence cleavage that is required for signal pathway activation. To deter- (block color; n ¼ 866) of coordinated ions (x axis values represent upper limit mine the mechanical stability of the NRR of the human Notch2 of histogram interval). Curve on extension histogram highlights the peaks receptor, AFM was performed on recombinant hN2-NRR (hN2 for LNR domains unfolding. Distinct HD domain unfolding not observed (ex- – pected extension 55 nm). Variations due to nonspecific attachments to the residues 1425 1672), comprising the LNR modules and HD do- hN2-NRR construct outside the polyK tag produce background features over main. AFM is used to apply force by pulling on single protein a large range of extensions. Curves on the force histogram highlight bimo- molecules at a constant loading rate, with the unfolding of a pro- dalality and therefore unfolding of two species. In the absence of ions, this tein domain corresponding to a peak in the observed force as the bimodal feature is lost and the force required for unfolding is reduced.

2of9 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1205788109 Stephenson and Avis Downloaded by guest on September 29, 2021 were repeatedly observed. Extension of LNRs to the full theore- as analysis of distances between defined secondary structure PNAS PLUS tical maximum (for 35–40 residues) of approximately 10.5–12 nm is elements. Fig. 4 shows differences in the force-extension profiles rarely observed due to the three disulphide bonds present in each produced in the presence and absence of ions (curves shown are LNR. Peaks in the saw-tooth profiles with larger force or extension representative of multiple obtained through repeated MD simula- features are attributed to the HD domain, for which full extension tions). Both Figs. 3 and 4 show snapshots of the unfolding protein to the theoretical maximum (55 nm) is also not observed. A slight structures associated with labeled transitions. Changes in the shortening of this domain is expected due to the presence of a sta- SAS of the S2 cleavage site provide a quantitative measure of the bilizing disulfide bond. However, the range of extensions observed accessibility of this region to metalloprotease cleavage. The largest also indicates some nonspecific attachment of the NHS linker to increase in the SAS of the S2 site occurs between 300 and 1,000 ps. lysine side chains in the HD domain. Histogram analysis of the When combined with simulation snapshots and the distance observed extensions demonstrates a unimodal non- distri- analyses (Fig. 3), it is evident that this increase results from the bution of the unfolding lengths of the NRR (Fig. 2B), where the removal of the A∶B linker from the S2 cleavage site. Correlation peak correlates to LNR unfolding between 5 and 15 nm (mode analysis of the SAS over this time and the distance between the 6.88 nm). The trailing tail of the distribution is due to the afore- A∶B linker and the S2 cleavage site shows strong correlation in mentioned attachment to lysine residues outside of the polyK-tag, all simulations performed (correlation coefficients between 0.76 resulting in the lack of a distinctive peak for the HD domain. and 0.80). Furthermore, the removal of the A∶B linker from the Forces required for NRR unfolding show a bimodal distribution cleavage site event corresponds to the first major feature (peak 1) (median ¼ 179 pN and 372 pN; Fig. 2C), consistent with the un- seen on a typical force extension curve (Fig. 4), which reaches a folding of two types of domain structure. Notably, the forces mea- force of approximately 375 pN at an extension of around 6.5 nm. sured fall within ranges observed for proteins with a load-bearing Further unfolding, specifically the removal of the S2 cleavage site mechanical role (37–39). The loading rate used for force measure- from the surrounding HD domain, causes smaller increases to the ments was 1 × 10 −7 N∕s (spring constant always within 10% of SAS at the S2 cleavage site while requiring similar levels of force 67 pN nm−1). As expected for unfolding of protein domains, force (peak 3, Fig. 4). The remaining peaks within Fig. 4 correspond to values associated with the recorded curves changed linearly with the the unfolding of the LNRB from the HD domain (peak 2) and the logarithm of the pulling speed (40) when this speed was altered. unraveling of the α3 helix (peak 4). Previous research has shown the removal of coordinated metal To determine whether exposure of the S2 site, through the re- ions leads to an open NRR structure with a more easily accessible moval of the A∶B linker, could allow access to metalloproteases,

S2 cleavage site (41) as well as allowing ligand-independent the partially unfolded NRR was manually docked into the active BIOPHYSICS AND

signaling activation (41, 42). Furthermore, there is evidence that site of TACE (44) (Fig. 5). The S2 site of the partially unfolded COMPUTATIONAL BIOLOGY the level of Notch signaling is sensitive to levels of extracellular construct fits nicely into the active site cleft of TACE with no calcium in vivo (43). It is thus pertinent to examine the influence steric hindrance from the surrounding structure. Hence, MD of coordinated metal ions on the mechanical unfolding of hN2- simulations combined with molecular demonstrate that, NRR. The differences observed in AFM experiments on protein after only small amounts of unfolding, the NRR structure is in a constructs containing and lacking (EDTA-treated) coordinated conformation providing access for metalloproteases known to ions are shown in Fig. 2B and C. Although a little difference is cleave it. observed in the unfolding lengths of these domains, the forces required for unfolding are dramatically lowered when coordi- Metal Ions Influence the Stability and Unfolding of the hN2-NRR. In nated ions are removed. Also, the bimodality of the force distri- AFM experiments, a notable reduction in forces for mechanical bution is lost when ions are removed, shifting the peaks of median unfolding of hN2-NRR was observed in the absence of metal 179 and 372 pN (ions present) to 65 pN (ions removed). The large ions, indicative of a major role for these ions (Ca2þ and Zn2þ) reduction in force required for unfolding and loss of the charac- in guiding the structural integrity of this region of Notch. The teristic distribution suggest the coordinated ions make a signifi- influence of metal ions was therefore examined further via simu- cant contribution to the structural integrity of the NRR. lation. When coordinated ions were removed from the MD simu- We also examined the influence of the disulfide bonds on the lations, four key changes were observed. Firstly, unfolding of the force-extension profiles by repeating the AFM experiments in the domains takes slightly longer when ions are removed. This first presence of DTT.Histogram analysis of the data (Fig. S1) shows a becomes apparent when the A∶B linker is pulled away from the marked increase in the extensions (median: wild type ¼ 18.9 nm, S2 cleavage site (Fig. 3). When the ions are removed, a delay is DTT treated ¼ 38.4 nm), while the force only slightly decreases seen for this unfolding event. Examination of the simulation out- (median: wild type ¼ 309.2 pN, DTT treated ¼ 238.2 pN). The put shows LNRA extending further when the calcium ions are increase in observed extension lengths correlates with lack of missing, causing a delay in the time it takes for the A∶B linker to observed extension of the LNRs, allowing us to more confidently be placed under force. Furthermore, when ions are present, the assign the histogram peaks in standard experiments to LNR and distance between the A∶B linker and S2 cleavage site stalls at HD domains, respectively. approximately 3.5 nm for 300 ps. This stall of unfolding occurs before the LNRB structure is removed from the HD domain and MD Simulations Show S2 Cleavage Site Exposure in hN2-NRR Corre- is caused by the lower extension (retention of more structure) of sponds to Removal of the Linker Region Between LNRA and LNRB. LNRA when ions are coordinated, leading to a more prominent To gain a greater understanding of the unfolding process that stall before a force magnitude is reached for LNRB unfolding to occurs when force is applied to the NRR, and to strengthen in- occur. An increase in time required for separation of LNRB from terpretation of the AFM data, MD simulations were performed the HD domain and for α3 helix unfolding and pulling away from using GROMACS (see Materials and Methods). Simulations were the β1 strand of the HD domain is also observed in the absence of carried out using the crystal structure of the hN2-NRR both in metal ions. These differences can also be linked to the shortened the presence and absence of coordinated ions [ time scale of the LNR domain unfolding when ions are present. (PDB) ID code 2OO4] (12), with application of a constant loading Secondly, the forces required for removing the A∶B linker rate (1 N∕s) on the atoms of the N-terminal residue (Cys1425) from the S2 cleavage site are affected by metal ions (Fig. 4). while the C-terminal residue (Thr1672) is positionally restrained. When coordinated ions are present, the force required for the The video output from the forced unfolding simulation is shown in removal of the A∶B linker from the S2 site forms one broad peak Movie S1. Fig. 3 shows the changes in solvent accessible surface at 375 pN spanning approximately 2.5 nm (peak 1, green) area (SAS) at the S2 cleavage site as the domains unfold, as well whereas, when these ions are removed, the barrier clearly sepa-

Stephenson and Avis PNAS Early Edition ∣ 3of9 Downloaded by guest on September 29, 2021 Fig. 3. Comparison of the changes in SAS of S2 cleavage site with the changes in distance between domains from MD simulations. (A) Differences in the distances between domains in the presence (dark) and absence (light) of coordinated ions do not affect the SAS of the S2 cleavage site (gray). (B) Screen shots of the corresponding changes in the NRR domain structure as unfolding occurs. Key changes are observed in the β5 strand housing the S2 cleavage site (blue; relevant side chains shown), and the LNR A∶B linker (pink; relevant side chains shown). Starting screenshot for each SAS increase shown semitransparent; arrows highlight change in structure. Data generated from GROMACS 4.5.3; images created in PyMol (version 1.3).

rates into two force peaks (peaks 1 and 1A, purple). These peaks Overall, removal of metal ions reduces the structural stability span approximately the same distance, although with a slight of the NRR, with a newly demonstrated role for zinc in stabilizing delay when ions are removed. The first of these two peaks cor- the LNR B∶HD and LNR C∶HD interface. The simulated responds to the unplugging of the S2 cleavage site and reaches a mechanical unfolding data concurs with the reduced forces for force of approximately 375 pN, comparable to the forces seen mechanical unfolding of hN2-NRR observed by AFM in the when ions are present. The second peaks at a force of around absence (versus presence) of metal ions. 450 pN and appears to be due to the methionine residue within the A∶B linker forming intermediate contacts with the side chains Mechanical Unfolding of hN2-NRR Allows Metalloprotease Cleavage at of the residues within the α3 helix. the S2 Site. Because simulation demonstrates that the S2 site is The third effect of metal ion removal, which in this case shows exposed early in the unfolding pathway of the NRR, experiments a change in force magnitude, occurs to the peak at approximately were performed to determine whether mechanical unfolding 10 nm extension (Fig. 4B; peak 2, with and without ions). This could lead to observation of cleavage at the S2 site. Initial experi- ments were performed to ensure that the TACE enzyme shows peak, which represents a barrier of approximately 200 pN in the activity for unfolded NRR in vitro. Firstly, cleavage assays were presence of ions, is reduced to a smaller undefined peak when performed using a quenched fluorescent peptide substrate, ions are absent. From examination of the screenshots, the force homologous to the S2 cleavage site of hN2. On incubation with event corresponds to the removal of LNRB from the HD domain. TACE, an increase in fluorescence was seen compared with the This event is thus particularly sensitive to the presence of metal peptide alone, showing cleavage of the peptide had been achieved ions during the simulated mechanical unfolding pathway. Inter- (Fig. 6A). Note that cleavage is specific, cleaving prior to the two ∶ estingly, a zinc ion is located between the B C linker and the HD Val residues as expected as determined by mass spectrum analysis domain. Although an effect of calcium ions on increasing the me- and N-terminal sequencing (Fig. S2). Analysis of the enzyme chanical stability of individual LNR modules can be rationalized specificity was then performed on the recombinant hN2-NRR and offered in explanation of differences in their unfolding time- structure. Because the S2 site is housed near the C terminus of scales (Fig. 3), the zinc ion is better positioned for a specific effect the protein, cleavage will remove the His tag from the protein, on disruption of the LNRB∶C linker–HD interaction. within an 18 amino acid (2.2 kDa) fragment. Given the small per- Finally, where coordinated ions are present, unfolding for the centage change in the molecular weight of N2-NRR that removal LNRC is not observed during the timescale of simulation. When of this fragment causes, changes in stained protein band migra- coordinated ions are absent, the LNRC begins to pull away from tion analyzed by SDS-PAGE are not conclusive. A Western blot the HD domain following β5 strand removal and α3 unfolding. was thus performed with antibodies raised against the C-terminal The partial unfolding of this domain when the coordinated zinc His6 tag (Fig. 6A, Inset) for protein samples that were folded or and calcium ions are absent again suggests a stabilizing role for denatured and treated or not treated with TACE. The denatured these ions. construct treated with TACE shows a loss of signal, compared

4of9 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1205788109 Stephenson and Avis Downloaded by guest on September 29, 2021 PNAS PLUS BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Fig. 4. Force-extension output from MD simulations comparing differences in the unfolding profile of the LNR-HD region in the presence and absence of ions. Force-extension graphs (B) comparing the absence (purple) and presence (green) of ions are staggered by 300 pN. The horizontal line dissecting the plots highlights the 200 pN point on both curves, raw data shown as well as a running average (period: 50). Screenshots for each highlighted peak are shown for simulations in the absence (A) and presence (C) of coordinated ions. Structure before unfolding peak is shown semitransparent and main features highlighted with color; arrows show key changes. Data generated from GROMACS 4.5.3; images created in PyMol (version 1.3).

with untreated denatured construct, and TACE treated “native” have taken up to 190 s (Fig. 6D). The methodology relies upon construct. In essence, TACE cleavage is specific to the hN2 S2 diffusion of the enzyme toward the clamped substrate within a cleavage site and cleaves only when the protein is in the dena- relatively short time frame at a lower than optimum temperature. tured form. The results confirm S2 site burial within the folded Given the nature of the experiment, the percentage of the recorded native protein and its exposure for TACE cleavage in the dena- extension curves that show loss of tension and tip retraction due to tured protein. The same experiments were performed using N2-NRR cleavage when TACE is injected is significant. Injection ADAM10, another metalloprotease known to cleave Notch in experiments were also performed with ADAM10. Although this vivo, and also demonstrated successful cleavage, albeit to a some- enzyme preparation is less active in vitro and is certainly more re- what lower degree (see Fig. S3). liant on incubation at 37 °C, it does show some ability to cleave Having established activity of TACE on hN2-NRR in vitro, during the force clamp (which could only be performed at room AFM experiments were set up to maintain a constant force of temperature), with 3.6% of force clamps showing cleavage events. 200 pN across the construct for 5 min. This level of force is suffi- Discussion cient to unfold LNRA and LNRB, and the S2 site should there- It is now accepted that activation of the Notch signaling pathway fore have become exposed. Lower forces were not possible in this relies on the S2 cleavage event (45). However, the mechanism by mode because of the difficulty in distinguishing attachments from which this cleavage is induced upon ligand binding has remained salt contacts. During this 5-min clamp at constant force, varying a subject for speculation. In its resting state, the hN2-NRR is in substances were injected in close proximity to the construct. an autoinhibited conformation (12), with a clear requirement for Essentially, if cleavage occurred during this clamp there would a change in structure to unmask the S2 site. A mechanotransduc- be a loss of tension between the tip and the surface of the sample, tion hypothesis is favored, in which the required conformational resulting in retraction of the tip (Fig. 6B). In experiments where change results from a mechanical force produced from the trans- buffer alone was injected, no cleavage events were observed endocytosis of the Notch extracellular domain into the ligand (Fig. 6C). When TACE was injected without the necessary metal presenting cell. Despite indirect evidence for this hypothesis, no ions, or when denatured TACE was injected, no cleavage events direct experimental evidence yet exists to demonstrate that a me- were observed. In comparison, when TACE and the necessary chanical force applied to a Notch molecule could actually unmask metal ions were injected, 46% of force clamps showed cleavage the S2 site. Here, we have shown, through the use of AFM and occurring. The majority of the TACE cleavage events occur MD simulations, that the S2 site of hN2-NRR is revealed with between 20 and 40 s after the point of injection; however, some minimal mechanical unfolding, allowing for cleavage at this site.

Stephenson and Avis PNAS Early Edition ∣ 5of9 Downloaded by guest on September 29, 2021 Fig. 5. Manual docking simulation of partially unfolded NRR in TACE active site. NRR from unfolding simulation (time point: 1,000 ps; following unfold- ing event 1 in Fig. 4C). (A) β5 strand housing the S2 cleavage site (light blue) fits snugly into the binding pocket of the TACE active site (moss green). Zinc binding residues and conserved catalytic residues are shown in lime green; coordinated zinc ion is shown in gray. (B) Rotation of 90° shows no steric hindrance from the HD domain (dark blue) at this early stage of unfolding. Docking performed with PyMol (version 1.3); TACE PDB ID code 1BKC (44).

Our research thus provides direct evidence on a molecular level to support the feasibility of a mechanotransduction mechanism for Notch signal activation. AFM experiments show that the forces required for NRR un- folding follow a bimodal distribution with peaks corresponding to Fig. 6. Cleavage experiments using TACE to determine unmasked S2 site unfolding forces with medians of 179 and 372 pN. The lower force specificity and whether mechanical force can allow for S2 cleavage. (A) Clea- peak is associated with unfolding of individual small LNR mod- vage is observed when TACE is incubated with hN2 S2 site fluorescent ules and the latter probably with the larger globular HD domain peptide. Increase in fluorescence over time after addition of TACE shows clea- (or a combination of this domain and LNRC, see below). The vage occurring at S2 site. Western blot (Inset) probed for the C-terminal recorded forces fall within the range required for unfolding of His-tag of recombinant protein shows a loss of signal in the denatured pro- immunoglobulin domains of mechanically stable titin-like pro- tein (d) containing TACE (þ) highlighting it’s specificity for the unfolded form teins (37–39), rather than nonmechanosensing proteins such as of the protein. Controls lacking TACE (−) or containing nondenatured protein n GFP and barnase (46, 47). The domains within the hN2-NRR ( ) show no cleavage. (B) Raw data from AFM cleavage experiments. The height of the AFM tip is measured over time; when protein is held, the region will thus clearly offer resistance to mechanical stimuli, tip is maintained close to the surface; however, when cleavage occurs tension in keeping with a mechanosensing role. across the tip is lost and retraction from the surface is observed. Point of TACE All-atom steered MD simulations were performed to provide injection is marked by arrowhead. (C) Graph showing percentage frequency greater insight into the mechanism behind the unfolding process of cleavage events when zinc chloride containing buffer (B, n ¼ 65), ADAM10 occurring when the Notch NRR is subject to force. Although (A, n ¼ 56), TACE in a zinc chloride buffer (Tþ, n ¼ 61), TACE without the ne- these simulations run at speeds of several orders of magnitudes cessary zinc ions (T−, n ¼ 54), and denatured TACE (dT, n ¼ 72) are injected. faster than in the AFM experiments, due to computational lim- (D) Graph showing time taken postinjection for cleavage events to occur x itations, and thereby give higher force measurements, such meth- when TACE was injected ( axis values represent the upper limit of the his- togram interval). ods can correctly predict mechanical unfolding pathways and certainly aid interpretation of experimental data (48, 49). The “ ” ∶ four domains of the NRR exhibit a largely but not entirely se- after the first main structure change that unplugs LNR A B β quential pattern of unfolding during MD simulation. Although linker from its position over the S2 site (located in the 5 strand “ ” ∶ the first two LNR modules unfold first, the β5 strand emerges of the HD domain). Removal of the plug formed by the A B from the HD structure, followed by further HD domain partial linker produces a large increase in the solvent accessibility of the β unraveling, without the prior requirement for LNRC unfolding. S2 cleavage site. Extraction of the 5 strand, housing the S2 site, Because LNRC unfolding is not observed in the period of our from the HD domain requires similar forces to those seen for the simulation, the forces required for the unfolding of this domain removal of the A∶B linker but produces only a minimal increase are much larger compared with the forces needed to unfold to the SAS, calling into question its importance in relieving auto- LNRA and LNRB. Therefore, we can more confidently conclude inhibition. Indeed, manual docking, performed after the removal that lower forces seen during LNR unfolding in AFM experi- of the A∶B linker, shows that the β5 strand bearing the S2 site ments correspond to the unfolding of the first two LNRs. Further- can be accommodated in the TACE active site cleft, without dis- more, larger force peaks in the AFM data, attributed to the HD ruption to the HD domain structure. Note that these findings are domain, might also be a combination of HD domain and LNRC different to those reported upon coarse grain simulation of the unfolding. Importantly, MD simulations reveal that the S2 clea- unfolding of the hN1-NRR (50), which show a perfect sequential vage site is exposed early in the mechanical unfolding pathway, removal of all three LNR domains from the HD structure and

6of9 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1205788109 Stephenson and Avis Downloaded by guest on September 29, 2021 further propose that some unraveling of the HD domain is po- the standard AFM unfolding of the hN2-NRR and on the basis PNAS PLUS tentially required for metalloprotease cleavage at S2. Although that MD simulations predicted that removal of LNRA and LNRB the unfolding pathways revealed by MD simulation differ slightly would expose the S2 site. Though AFM is a sensitive approach, it for N1 versus N2-NRR, it is difficult to be certain whether this is unfeasible to control it to the level where only LNRA and the difference is a reflection of the different simulation methods used A∶B linker are unraveled. The selected force clamp would ensure or the different protein subject. Notably, though, the hN1-NRR removal of at least the first two LNRs when metalloprotease is does not contain a zinc ion located between the HD domain and injected; a reasonable compromise. The frequency of hN2-NRR the LNRB-C linker region. Both AFM and the MD simulations cleavage events observed was significant in the given experimen- on hN2-NRR demonstrate metal ion-dependent structural tal conditions and directly links force application to exposure and stability of the NRR region. Moreover, our simulations highlight cleavage of the S2 site. a role for the zinc ion in increasing the stability of the HD Interestingly, TACE cleaved the S2 cleavage site of hN2-NRR domain∶LNRC interaction through its interactions with residues more readily than ADAM10 within all experiments mentioned from the B∶C linker, LNRC, and the HD domain, thereby influ- in this study. Certainly, ADAM10 is more dependent than TACE encing the unfolding pathway. To date, there is a lack of experi- on incubation at 37 °C, and this is likely the main factor for a mental data for N1-NRR unfolding, though a preliminary AFM lower percentage of cleavage occurring in both the Western blot study on the mouse N1-NRR has recently been published that and AFM experiments (performed at room temperature). reports similar sizes of unfolded LNR modules to those we report However, because recent research has highlighted an essential here for hN2 but in addition to sequential unfolding of these, role for ADAM10 in ligand-dependent activation (14), whereas apparently also observes complete unfolding of the HD domain ADAM10 and TACE have redundant roles in ligand independent (51). Although these data might support a protein sequence basis activation (14, 56), we cannot completely exclude the possibility for a slight difference in simulated unfolding pathway for the two that the AFM force-clamp setup may be a less accurate reflection NRRs, the preliminary nature of the N1-NRR AFM study, its of the natural ligand-dependent forced unfolding process. high dependence on the simulation data (50) for interpretation All the data presented show that exposure of the S2 site occurs of extension curves, and its use of different methodology to our early on the hN2-NRR unfolding pathway at force levels that cor- study prevent a thorough comparison at this stage. relate reasonably well with the adhesion strength between Notch We suggest a mechanism whereby autoinhibition is relieved and its ligand (21, 57) and with ligand endocytic force. We pro- and S2 cleavage can feasibly occur within hN2-NRR following the pose that the first and most critical force barrier in NRR unfold-

removal of the A∶B linker. Interestingly, analysis of S2 site expo- ing (A∶B linker removal) is the mechanosensing event within the BIOPHYSICS AND ∶

sure in the hN1-NRR (41) by hydrogen exchange mass spectro- Notch extracellular domain. Although A B removal is critical, COMPUTATIONAL BIOLOGY metry also deduced that detachment of LNRA and LNRB (upon the NRR is actually quite “mechanoresistant” with respect to removal of metal ions) caused sufficient exposure of the S2 site full unfolding of the individual domains, especially upon metal for metalloprotease cleavage, with no requirement for disruption ion coordination. This mechanoresistance could feasibly enable to the structural integrity of the HD domain. The force exerted ready restoration of Notch structure should other factors release upon the NRR during the transendocytosis process in vivo is the bound Notch ligand. The major contribution of metal ion co- unknown, but, if our proposed mechanism is to hold, it must be ordination to NRR mechanoresistance could have physiological sufficient to remove the A∶B linker. Our hN2-NRR AFM data significance also. For example, the level of Notch signaling has show full unfolding of LNRA and B occurs at forces a little less already been linked to extracellular concentrations of calcium than 200 pN. Endocytosis has previously been observed to pro- (43), and there is an additional possibility that exposure to a drop duce uptake forces of around 20–80 pN (at a similar loading rate in pH such as occurs during endocytosis could affect metal coor- to that used here) (52). Recently, epsin-dependent clathrin- dination and rate of cleavage at S2. The Notch NRR in the full- mediated endocytosis of the mammalian Notch ligand, Delta- length receptor is preceded by EGF-like repeats, which would be like1, has been shown to generate a 10-pN net force on a bead- predicted to offer considerable mechanical stability also, based tethered N-terminal fragment of rat Notch1 (excluding the NRR) on the precedence of β-sheet dominated structures to show the (53). Although these measured endocytic force levels may appear highest mechanical stability (37–39). Future work to test the in- low, no experimental single-molecule system has yet mimicked fluence of these preceding EGF-like repeats would, however, be the ligand-induced Notch signaling event between two cells. of interest. A further issue to explore is whether cleavage at S1 is The tethering of the NRR to a solid substrate in our experimental required for S2 cleavage, for which contradictory data exist (23, setup, rather than a membrane (or a movable bead), may indeed 58, 59), probably due to differences in organism and type of inflate recorded forces in AFM experiments compared with those Notch receptor. Here, we show protease accessibility to S2 upon that occur in vivo. It is also the case that other signaling processes forced unfolding of hN2-NRR that possesses an intact S1 site. are known that entail internalization of a full transmembrane This result would concur with the observation that S1-resistant ligand protein by transendocytosis (54), indicative of endocytic Notch2 retains signaling competency (60). S1-resistant Notch1 force reaching a level sufficient to remove a protein entirely from has reduced signaling competency, however, and similar forced a membrane. Recorded forces for removal of helices from mem- unfolding work with this receptor may yield different observa- branes are in excess of 200 pN (55). Overall, the forces recorded tions. The influence of S1 cleavage (and, indeed, other factors) here for LNR unfolding could fit with an endocytic mechanism on mechanical unfolding and S2 exposure would be best tested for forced exposure of the S2 site in Notch, especially since we using a single-molecule experimental setup that tethered the propose that removal of the A∶B linker is sufficient for cleavage. NRR (of different Notch receptor types) to a membrane. Current It is still possible that some local unfolding of the β5 strand may work is directed toward these technically challenging experi- further increase the cleavage propensity and Notch activation, ments, ideally with detection of Notch signal transmission to a especially if the stability of this region is affected, as is thought cell nucleus after force activation. Although questions, therefore, to be the case in T-cell acute lymphoblastic leukemia lymphoma remain concerning Notch mechanotransduction, we have com- causing mutations that occur within the HD domain of human bined experimental and computational modeling approaches to Notch1 (9). reveal new direct evidence for, and insight into, the molecular A vital part of our study is that we have been able to show mechanism of the process. Further methodological developments experimentally that injection of metalloproteases during forced will help answer the remaining questions, with potential for ther- unfolding causes hN2-NRR cleavage at the S2 site. The force apeutic applications in Notch-related disease, as well as enhance clamp, 200 pN, was selected based on the forces recorded during exploration of mechanical force in other signaling pathways.

Stephenson and Avis PNAS Early Edition ∣ 7of9 Downloaded by guest on September 29, 2021 Materials and Methods the level of features occurring due to interactions between the functiona- Protein Expression and Purification. Recombinant NRR was produced as a GST- lized slide and tip. Surface adhesion contacts appearing at the foot of the fusion protein with N-terminal poly-lysine (Lys3) and C-terminal hexahistidine retraction slope, distinct from unfolding features observed when protein is (His6) tags through expression in T7 Express Escherichia coli cells (New Eng- present, could thus be identified. Attachment controls were also performed land Biolabs). Cells were lysed by addition of lysozyme and three rounds of using competing ions to disrupt Ni2þ-His interactions at the tip. freeze thaw. Protein was purified from soluble lysate through incubation with glutathione beads (GE Healthcare), cleaving the GST-tag to release AFM Experiments with Cleavage. Functionalization was performed as above; the protein. To gain further purity, the protein was denatured and purified force experiments were performed on the JPK CellHesion200 in PBS at a load- 2þ further with ion-exchange (Ni -His; His-Trap™ HP columns) and size exclu- ing rate of approximately 0.6 × 10−7 N∕s (spring constant approximately sion chromatography (S-200 Superdex) (columns from GE Healthcare). Puri- 67 pN∕nm; velocity 850 nm∕s). A 2-s surface delay was used, before a force fied protein was refolded in a redox buffer (containing 5 mM cysteine, 1 mM clamp of 200 pN was maintained for 300 s (5 min). At approximately 30 s, cystine, and 50 mM CaCl2) before structural characterization was performed 1.32 μL of solution was injected. Solutions injected were buffer (B; 25 mM 1 1 to determine correct structural conformation (1D- H NMR, 2D H-NOESY Tris-HCl+2.5 μM zinc chloride, pH 9.0), ADAM10 (A; 50 ng∕μL ADAM10 in buf- NMR, CD, UV resonance Raman spectroscopy). fer), TACE with required ions (Tþ; 50 ng∕μL TACE in buffer), TACE lacking required ions (T−; 50 ng∕μL TACE in 25 mM Tris-HCl, pH 9.0), denatured TACE Fluorescent Peptide Cleavage. Quenched peptide (MCA-GSYPLVSVVSE-Dap (dT; 50 ng∕μL TACE in buffer, following three rounds of freeze thaw to de- μ (DNP)-SLT-NH2; Generon) (10 mM) in 25 mM Tris-HCl, 2.5 M ZnCl2,pH9.0 nature (inactive in fluorescent peptide cleavage experiments). was incubated for 5 min with 0.1 ng∕μL TACE (mature soluble extracellular region, R&D Systems) at room temperature or 0.1 ng∕μL ADAM10 (R&D MD Simulations. MD simulations were performed using GROMACS (61) ver- Systems) at 37 °C before being excited at 320 nm. Absorption spectra were sion 4.5.3 on hN2-NRR (PDB ID code 2OO4) with and without coordinated recorded (320–600 nm) every 5 min to a total of 90 min at either room tem- ions present. Protein termini were protonated, and titratable amino acids perature (TACE) or 37 °C (ADAM10). The absorption peak at 405 nm was used were assigned their canonical state at physiological pH. The se- to follow change in activity over time. lected was the GROMOS96 53A6 parameter set (62). Short-range interaction cutoff was set to 1.4 nm and long-range electrostatics were calculated with Mass Spectrometry of Peptide Cleavage. The same quenched peptide the particle mesh Ewald algorithm (63, 64). Dispersion correction was applied (0.24 mM) in 25 mM Tris-HCl, 2.5 μM ZnCl2, pH 9.0 was incubated for 24 h to energy and pressure terms to account for truncation of van der Waals with 0.78 μg TACE (mature soluble extracellular region, R&D Systems) at room temperature. The reaction was then purified using a ZipTip (Merck terms. Periodic boundary conditions were applied in all directions. Millipore) eluted into 5 μL 50% Acetonitrile, 0.1% Trifluoroacetic acid. Mass Protein (coordinates) was placed in a three-dimensional box (dimensions: 10 × 10 × 50 spectrometry analysis was performed on a Bruker Ultraflex II TOF/TOF using nm) of 100 nM NaCl in simple point charge water (65), including 1 μL eluted material in a 1∶1 ratio with α-Cyano-4-hydrozycinnamic acid ma- neutralizing counterions. Steepest descent energy minimization was per- trix (10 mg∕mL in 50% ethanol, 50% acetonitrile). formed followed by a two-step equilibration, with position restraints applied to heavy atoms. Equilibration step one simulated 100 ps under the NVT en- Western Blot Cleavage. Denatured (incubated overnight in 8 M urea) and semble (maintaining a constant number of particles, volume, and tempera- wild-type recombinant hN2 Lys3-NRR-His6 was diluted 1 in 100 with a clea- ture). Temperature was maintained at 310 K (37 °C) by coupling protein and vage buffer containing zinc ions (25 mM Tris-HCl, 2.5 μM ZnCl2, pH 9.0) and nonprotein atoms to separate temperature coupling baths [Berendsen weak incubated at room temperature for 24 h with either 0.1 ng∕μL TACE or coupling method (66)]. Equilibration step two simulated 100 ps under the 0.1 ng∕μL ADAM10. Results were analyzed by Western blot. The blot on NPT ensemble, maintaining a constant isotropic pressure of 1.0 bar (weak Hybond C (GE Healthcare) was blocked with 5% milk powder in TBS+Tween coupling). All position restraints were then removed, except for those on (2 h), incubated with HRP conjugated mouse anti-His6 antibody (Invitrogen) the atoms of the C-terminal residue (Thr1672), which was used as an immo- in blocking solution (16 h), washed, and viewed by using ECL Plus Western bile reference for the pull simulation. For each simulation, the atoms of the Blotting kit (Pierce) and exposing to film. N-terminal residue (Cys1425) were pulled along the z axis at a loading rate of approximately 1 N∕s (spring constant: 1.66 × 10−9 N∕nm; velocity: Standard AFM Experiments. Gold-coated AFM tips were functionalized with 1 × 109 nm∕s. These simulations used the Nosé–Hoover thermostat (67, 2þ Ni -NTA by incubating overnight with a 0.01 mM NTA-alkanethiol∕ 68) and the Parrinello–Rahman barostat (69, 70). Pearson’s linear correlation 0.04 mM EG3-alkanthiol solution (obtained from Prochimia and Nanoscience coefficient was calculated across five of the obtained simulation data sets Instruments, respectively), followed by 1-h incubation with nickel sulphate using MATLAB R2010a. (Sigma Aldrich). Gold-coated AFM slides were functionalized by incubating overnight with a 0.1 mM NHS-alkanethiol∕0.04 mM EG3-alkanthiol solution ACKNOWLEDGMENTS. We thank the late Anne-Marie Buckle for a collabora- (both obtained from Nanoscience Instruments), followed by a 1-h incubation tion that generated the ideas and confidence to initiate this work. We thank with protein solution in PBS, and 1 M ethanolamine (Sigma Aldrich) incuba- Steven Marsden for his assistance in the Bionanotechnology facility in the tion for 40 min. Force experiments were performed on a Nanoscope V con- Faculty of Life Sciences, Andrew Doig for his advice during the course of −7 troller in PBS buffer at a loading rate of approximately 1 × 10 N∕s (spring the project, Kathryn Blount for reading and improving the manuscript, constant approximately 67 pN∕nm; velocity approximately 1;600 nm∕s), with and the GROMACS Users List for their assistance with our queries. This work a 2-s surface delay to ensure nickel-His coordination (based on previous ex- was supported by the Biotechnology and Biological Sciences Research Council periments). Controls were performed without protein present to determine (UK) via a doctoral training award to N.L.S. (Award BB/D526561/1).

1. Tien AC, Rajan A, Bellen HJ (2009) A Notch updated. J Cell Biol 184:621–629. 11. Sanchez-Irizarry C, et al. (2004) Notch subunit heterodimerization and prevention of 2. Andersson ER, Sandberg R, Lendahl U (2011) Notch signalling: Simplicity in design, ligand-independent proteolytic activation depend, respectively, on a novel domain versatility in function. Development 138:3593–3612. and the LNR repeats. Mol Cell Biol 24:9265–9273. 3. Artavanis-Tsakonas S, Rand MD, Lake RJ (1990) Notch signalling: Cell fate control and 12. Gordon WR, et al. (2007) Structural basis for autoinhibition of Notch. Nat Struct Mol – signal integration in development. Science 284:770–776. Biol 14:295 300. 4. Bray SJ (2006) Notch signaling: A simple pathway becomes complex. Nat Rev Mol Cell 13. Brou C, et al. (2000) A novel proteolytic cleavage involved in Notch signalling: The role – Biol 7:678–689. of the disintegrin-metalloprotease TACE. Mol Cell 5:207 216. 14. Bozkulak EC, Weinmaster G (2009) Selective use of ADAM10 and ADAM17 in Notch 5. Meier-Stiegen F, et al. (2010) Activated Notch1 target genes during embryonic cell signalling. Mol Cell Biol 29:5679–5695. differentiation depend on the cellular context and include lineage determinants 15. Fehon RG, et al. (1990) Molecular interactions between the protein products of the and inhibitors. PLoS One 5:e11481. neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila. Cell 6. Rampal R, Luther KB, Haltiwanger RS (2007) Notch signalling in normal and disease 61:523–534. states: Possible therapies related to glycosylation. Curr Mol Med 7:427–445. 16. Fleming RJ (1998) Structural conservation of Notch receptors and ligands. Semin Cell 7. Weng AP, et al. (2004) Activating mutations of NOTCH1 in human T cell acute lympho- Dev Biol 9:599–607. – blastic leukemia. Science 306:269 271. 17. Gordon WR, et al. (2009) Structure of the Notch1-negative regulatory region: Implica- 8. Louvi A, Arboleda-Velasquez JF, Artavanis-Tsakonas S (2006) CADASIL: A critical look at tions for normal activation and pathogenic signalling in T-ALL. Blood 113:4381–4390. a Notch disease. Dev Neurosci 28:5–12. 18. Rebay I, et al. (1991) Specific EGF repeats of Notch mediate interactions with Delta and 9. Aster JC, Blacklow SC, Pear WS (2011) Notch signalling in T-cell lymphoblastic leukae- Serrate: Implications for Notch as a multifunctional receptor. Cell 67:687–699. mia/lymphoma and other haematological malignancies. J Pathol 223:262–273. 19. Xu A, Lei L, Irvine KD (2005) Regions of Drosophila Notch that contribute to ligand 10. Koch U, Radtke F (2010) Notch signalling in solid tumors. Curr Top Dev Biol 92:411–455. binding and the modulatory influence of Fringe. J Biol Chem 280:30158–30165.

8of9 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1205788109 Stephenson and Avis Downloaded by guest on September 29, 2021 20. Cordle J, et al. (2008) A conserved face of the Jagged/Serrate DSL domain is involved in 47. Dietz H, Rief M (2004) Exploring the energy landscape of GFP by single-molecule me- PNAS PLUS Notch trans-activation and cis-inhibition. Nat Struct Mol Biol 15:849–857. chanical experiments. Proc Natl Acad Sci USA 101:16192–16197. 21. Ahimou F, Mok LP, Bardo B, Wesley C (2004) The adhesion force of Notch with Delta 48. West DK, Brockwell DJ, Olmsted PD, Radford SE, Paci E (2006) Mechanical resistance of and the rate of Notch signaling. J Cell Biol 167:1217–1229. proteins explained using simple molecular models. Biophys J 90:287–297. 22. Parks AL, Klueg KM, Stout JR, Muskavitch MA (2000) Ligand endocytosis drives recep- 49. Sotomayor M, Schulten K (2007) Single-molecule experiments in vitro and in silico. tor dissociation and activation in the Notch pathway. Development 127:1373–1385. Science 316:1144–1148. 23. Nichols JT, et al. (2007) DSL ligand endocytosis physically dissociates Notch1 heterodi- 50. Chen J, Zolkiewska A (2011) Force-induced unfolding simulations of the human – mers before activating proteolysis can occur. J Cell Biol 176:445 458. Notch1 negative regulatory region: Possible roles of the heterodimerization domain 24. Sun X, Artavanis-Tsakonas S (1997) Secreted forms of DELTA and SERRATE define in mechanosensing. PLoS One 6:e22837. – antagonists of Notch signaling in Drosophila. Development 124:3439 3448. 51. Dey A, Szoskiewicz R (2012) Complete noise analysis of a simple force spectroscopy 25. Varnum-Finney B, et al. (2000) Immobilization of Notch ligand, Delta-1, is required for AFM setup and its applications to study nanomechanics of mammalian Notch 1 induction of notch signalling. J Cell Sci 113:4313–4318. protein. Nanotechnology 23:175101. 26. Vas V, Szilágyi L, Pálóczi K, Uher F (2004) Soluble Jagged-1 is able to inhibit the 52. Shan Y, et al. (2011) Recording force events of single quantum-dot endocytosis. Chem function of its multivalent form to induce hematopoietic stem cell self-renewal in Commun 47:3377–3379. a surrogate in vitro assay. J Leukoc Biol 75:714–720. 53. Meloty-Kapella L, Shergill B, Kuon J, Botvinick E, Weinmaster G (2012) Notch ligand 27. Jaalouk DE, Lammerding J (2009) Mechanotransduction gone awry. Nat Rev Mol Cell endocytosis generates mechanical pulling force dependent on dynamin, epsins, and Biol 10:63–73. actin. Dev Cell 22:1299–1312. 28. White CR, Frangos JA (2007) The shear stress of it all: The cell membrane and mechan- ochemical transduction. Philos Trans R Soc Lond B Biol Sci 362:1459–1467. 54. Cagan RL, Krämer H, Hart AC, Zipursky SL (1992) The bride of sevenless and sevenless – 29. Lecuit T, Lenne P-F (2007) Cell surface mechanics and the control of cell shape, tissue interaction: Internalization of a transmembrane ligand. Cell 69:393 399. patterns and morphogenesis. Nat Rev Mol Cell Biol 8:633–644. 55. Oesterhelt F, et al. (2000) Unfolding pathways of individual Bacteriorhodopsins. – 30. Liu J, Kaksonen M, Drubin DG, Oster G (2006) Endocytic vesicle scission by lipid phase Science 288:143 146. boundary forces. Proc Natl Acad Sci USA 103:10277–10282. 56. van Tetering G, et al. (2009) Metalloprotease ADAM10 is required for Notch1 site 2 31. Friedland JC, Lee MH, Boettinger D (2009) Mechanically activated integrin switch cleavage. J Biol Chem 284:31018–31027. controls a5b1 function. Science 323:642–644. 57. Shergill B, Meloty-Kapella L, Musse AA, Weinmaster G, Botvinick E (2012) Optical 32. Sukharev S, Anishkin A (2004) Mechanosensitive channels: What can we learn from tweezers studies on Notch: Single-molecule interaction strength is independent of ‘simple’ model systems? Trends Neurosci 27:345–351. ligand endocytosis. Dev Cell 22:1313–1320. 33. Sawada Y, et al. (2006) Force sensing by mechanical extension of the Src family kinase 58. Kidd S, Lieber T (2002) Furin cleavage is not a requirement for Drosophila Notch func- substrate p130Cas. Cell 127:1015–1026. tion. Mech Dev 115:41–51. 34. Gudi S, Nolan JP, Frangos JA (1998) Modulation of GTPase activity of G proteins by fluid 59. Pratt EB, et al. (2011) The cell giveth and the cell taketh away: An overview of Notch shear stress and phospholipid composition. Proc Natl Acad Sci USA 95:2515–2519. pathway activation by endocytic trafficking of ligands and receptors. Acta Histochem 35. Carrion-Vazquez M, Oberhauser AF, Fernandez JM (1999) Mechanical and chemical 113:248–255. unfolding of a single protein: A comparison. Proc Natl Acad Sci USA 96:3694–3699. 60. Gordon WR, et al. (2009) Effects of S1 cleavage on the structure, surface export, and 36. Steward A, Toca-Herrera JL, Clarke J (2002) Versatile cloning system for construction of signaling activity of human Notch1 and Notch2. PLoS One 4:e6613. multimeric proteins for use in atomic force microscopy. Protein Sci 11:2179–2183. 61. Hess B, Kutzner C, van der Spoel D, Lindahl E (2008) GROMACS 4: Algorithms for highly BIOPHYSICS AND 37. Rief M, Gautel M, Oesterhelt F, Fernandez JM, Gaub HE (1997) Reversible unfolding of efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput – COMPUTATIONAL BIOLOGY individual titin immunoglobulin domains by AFM. Science 276:1109 1112. 4:435–447. 38. Oberhauser AF, Badilla-Fernandez C, Fernandez JM (2002) The mechanical hierarchies 62. Oostenbrink C, Villa A, Mark AE, Van Gunsteren WF (2004) A biomolecular force – of fibronectin observed with single-molecule AFM. J Mol Biol 319:433 447. field based on the free enthalpy of hydration and salvation: The GROMOS force-field 39. Bullard B, et al. (2006) The molecular elasticity of the insect flight muscle proteins parameter sets 53A5 and 53A6. J Comput Chem 25:1656–1676. projectin and kettin. Proc Natl Acad Sci USA 103:4451–4456. 63. Darden T, York D, Pedersen L (1993) Particle mesh Ewald: An n•log(n) method for 40. Evans E, Ritchie K (1999) Strength of a weak bond connecting flexible polymer chains. Ewald sums in large systems. J Chem Phys 98:10089–10092. Biophys J 76:2439–2447. 64. Essmann U, et al. (1995) A smooth particle mesh Ewald method. J Chem Phys 41. Tiyanont K, et al. (2011) Evidence for increased exposure of the Notch 1 metallo- 103:8577–8593. protease cleavage site upon conversion to an activated conformation. Structure 65. Berendsen HJC, Postma JPM, van Gunsteren WF, Hermans J (1981) Intermolecular 19:546–554. – 42. Rand MD, et al. (2000) Calcium depletion dissociates and activates heterodimeric Forces, ed B Pullman (Reidel, Dordrecht, The Netherlands), pp 331 342. Notch receptors. Mol Cell Biol 20:1825–1835. 66. Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR (1984) Molecular- – 43. Raya A, et al. (2004) Notch activity acts as a sensor for extracellular calcium during dynamics with coupling to an external bath. J Chem Phys 81:3684 3690. vertebrate left-right determination. Nature 427:121–128. 67. Nosé SA (1984) A unified formulation of the constant temperature molecular – 44. Maskos K, et al. (1998) Crystal structure of the catalytic domain of human tumor dynamics methods. J Chem Phys 81:511 519. necrosis factor-alpha-converting enzyme. Proc Natl Acad Sci USA 95:3408–3412. 68. Hoover WG (1985) Canonical dynamics: Equilibrium phase-space distributions. Phys 45. Kopan R, Ilagan MX (2009) The canonical Notch signalling pathway: Unfolding the Rev A 31:1695–1697. activation mechanism. Cell 137:216–233. 69. Nosé S, Klein ML (1983) Constant pressure molecular dynamics for molecular systems. 46. Best RB, Li B, Steward A, Daggett V, Clarke J (2001) Can non-mechanical proteins with- Mol Phys 50:1055–1076. stand force? Stretching barnase by atomic force microscopy and molecular dynamics 70. Parrinello M, Rahman A (1981) Polymorphic transitions in single crystals: A new simulation. Biophys J 81:2344–2356. molecular dynamics method. J Appl Phys 52:7182–7190.

Stephenson and Avis PNAS Early Edition ∣ 9of9 Downloaded by guest on September 29, 2021