100 101 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Understanding how cells communicate could help treat cancer Related publication: Zeronian, M. R., Klykov, O., Portell i deMontserrat, J., Konijnenberg, M. J., Gaur, A., Scheltema, R. A., & Janssen, B. J. C. Notch–Jagged signaling complex defined by an interactionmosaic. Proceedings of the National Academy of Sciences 118, (2021). DOI: 10.1073/ pnas.2102502118 Publication keywords: Receptor; Ligand; Complex; Cell signalling; SAXS; Structure; X-ray diffraction C ommunication between cells is essential for the development and stability of tissues and prevents diseases, including cancer. The Notch and Jagged transmembrane proteins interact to regulate communication between cells in all multicellular animals. Defining these interactions is critical to understanding Notch-associated disorders. However, while structural studies have focused on short regions of both proteins, it remained unclear how their entire extracellular domains collectively engage to activate signalling. Researchers used Small-Angle X-Ray Scattering (SAXS) measurements on the High-throughput SAXS beamline (B21) to probe the low- resolutionpropertiesof theNotch1andJagged1ectodomain. SAXS turnedout tobeveryuseful toshowthat theproteinshaveconformational flexibility. They also used the Macromolecular Crystallography beamline I03 for X-ray diffraction on crystals of a smaller Notch1 segment. Their results identified several unreported, interacting regions in the Notch1–Jagged1 full extracellular complex. The Notch1 and Jagged1 ectodomains are flexible and not fully extended. Notch1 and Jagged1 interact through more sites than previously thought, and regions of importance, i.e. , for Notch1 activation, are in direct contact in the Notch1-Jagged1 complex. Knowing which parts in the Notch1-Jagged1 complex are interacting will help in the design of tools to interfere with this interaction. This may be useful for new therapeutics in the fight against cancer. This interaction network redefines our knowledge on Notch activation and provides avenues for therapeutic advances. The cell-surface receptor Notch1 is an important factor in the communication between cells and controls the development and function of tissues. Interactions between Notch1 and the ligand Jagged1 regulates cell differentiation inmulticellular animals including humans 1 . Notch1 dysfunction often leads to developmental disorders and cancers, and as such, Notch1 is a sought-after therapeutic target 2 . Notch1 signaling is initiated by Jagged1 binding. Jagged1 binding triggers a conformational change in Notch1, exposing sites for proteolytic processing in the C-terminal membrane proximal domain, releasing the Notch1 intracellular segment that can then travel to the nucleus to regulate transcription of target genes 3 . Jagged1 interaction can also inhibit Notch1 signaling when the proteins are expressed on the same cell. How Notch1 and Jagged1 interact is not entirely resolved. Notch1 and Jagged1 are large proteins, with 40 and 19 domains on the extracellular side, respectively, of which epidermal growth factor (EGF) repeats make up a large part. The extracellular segments are believed to be conformationally flexible, although this has only been experimentally determined for a few smaller portions in the extracellular segments 4 .Work during the past three decades has focused on two sites in this signalling pair, the EGF8-EGF12 region in Notch1 and the C2-EGF3 region in Jagged1. Recent crystal structure data has shown that these segments interact in an antiparallel fashion 5 . How other sites contribute to Notch1-Jagged1 interaction in the context of the full extracellular segments is unknown. Also, the coupling of Jagged1 binding to Notch1 EGF8-EGF12 and the subsequent conformational change in the Notch regulatory region (NRR) separated by 24 EGF repeats is not resolved. Structural studies on the full ectodomains have been hampered due to their large size, extensive N- and O-linked glycosylation, numerous disulfide bonds – 119 in Notch1 – and concomitant difficulty in producing purified samples. By using human embryonic kidney (HEK293) cell suspension culture at multi-litre scale it was possible to obtain sufficient full ectodomain Notch1 and Jagged1 sample for Size Exclusion Chromatography Small Angle X-ray Scattering (SEC-SAXS) experiments. SEC-SAXS on these purified samples, measured at Diamond Light Source beamline B21 and ESRF beamline BM29, revealed that the ectodomain conformations of Notch1 (Notch1 fe ) and Jagged1 (Jagged1 fe ) are flexible and not fully extended (Fig. 1). The maximum dimension ( D max ) of the Notch1 ectodomain is 380 Å, whereas that of Jagged1 is 240 Å. If both proteins would be fully extended, as schematically drawn in Fig. 1 (left), the D max would have been 1,027 Å for Notch1 fe and 585 Å for Jagged1 fe . Considering the flexibility and size of the samples, more detailed structural studies on the full ectodomains were predicted to be challenging. Instead, smaller segments were analysed by X-ray diffraction and batch SAXS. A crystal structure of Notch1 NRR, determined from 2.1 Å diffraction data collected at Diamond beamline I03, revealed a dimer that is compatible with Notch1 dimerisation as a full-length protein on the cell surface (Fig. 2a). The dimerisation of Notch1 NRR was confirmed in solution by Size Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS). Batch SAXS analysis, at Diamond beamline B21, of two smaller Jagged1 segments at a range of concentrations showed both segments do not change their oligomeric state in solution (Fig. 2b). The four-domain EGF8-11 segment of Jagged1 has conformational flexibility whereas the Jagged1 cysteine-rich domain (CRD) is globular and compact. The flexibility and the size of the Notch1 and Jagged1 ectodomains may enable them to interact through multiple sites. Cross-Linking Mass-Spectrometry (XL-MS) analysis on the Notch1 fe – Jagged1 fe complex indicated that the C-terminal region of Notch1 fe , centred on the EGF33-NRR segment, played a role in interaction with three regions in Jagged1; C2-EGF3, EGF8-11 and CRD (Fig. 3a). In addition, a multitude of crosslinks in Jagged1 fe suggested substantial intramolecular interactionswithin the Jagged1 ectodomain. The proximity of Notch1 EGF33-NRR to Jagged1 C2-EGF3 in the complex is remarkable as this could implicate amore direct role for Jagged1 binding to Notch1 NRR activation. Quantitative interaction experiments, i.e. Surface Plasmon Resonance (SPR) and Microscale Thermophoresis (MST) of smaller targeted segments, confirmed the intermolecular and intramolecular interactions indicated by the XL-MS analysis. In addition, direct interaction of Notch1 EGF8-EGF13 and Notch1 NRR showed that these two functionally important segments are likely much closer within Notch1 than previously thought. These data, on conformational flexibility and a mosaic of interaction sites, suggest that Notch1 and Jagged1 can interact intimately, and that the interaction mode is, most likely, dependent on their setting on the same cell (in cis ) or on opposing cells (in trans ) (Fig. 3b). The identification of previously unreported Notch1 and Jagged1 interactions sites opens the possibility for the development of new therapeutics that target these sites to prevent Notch1 activation in cancers. References: 1. Bray, S. J. Notch signalling in context. Nature Reviews Molecular Cell Biology 17, 722–735 (2016). DOI: 10.1038/nrm.2016.94 2. Aster, J. C. et al. The varied roles of Notch in cancer. Annual Review of Pathology: Mechanisms of Disease 12, 245–275 (2017). DOI: 10.1146/ annurev-pathol-052016-100127 3. Kovall, R. A. et al. The canonical Notch signaling pathway: structural and biochemical insights into shape, sugar, and force. Developmental Cell 41, 228–241 (2017). DOI: 10.1016/j.devcel.2017.04.001 4. Weisshuhn, P. C. et al. Non-linear and flexible regions of the human Notch1 extracellular domain revealed by high-resolution structural studies. Structure 24, 555–566 (2016). DOI: 10.1016/j.str.2016.02.010 5. Luca, V. C. et al. Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity. Science 355, 1320–1324 (2017). DOI: 10.1126/science.aaf9739 Funding acknowledgement: This project was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme with grant agreement No. 677500 (to B.J.C.J.); the research programme TA with project number 741.018.201 (to R.A.S.), which is partly financed by the Dutch Research Council (NWO); and the European Union Horizon 2020 programme INFRAIA project Epic-XS project 823839 (to R.A.S.). This work benefited from access to the Amsterdam NKI, an Instruct-ERIC centre, with financial support provided by Instruct-ERIC (PID 10025). This work has been supported by iNEXT (PID 6764), funded by the Horizon 2020 programme of the European Union. Corresponding author: Dr. Bert Janssen, Utrecht University,
[email protected] Soft CondensedMatter Group Beamline B21 (andMacromolecular Crystallography Group Beamline I03) Figure 2: Segments of the Notch1 and Jagged1 ectodomain have different properties; (a) Crystal structure of Notch1NRR, consisting of three small Lin12/Notch repeats and a heterodimerisation domain, reveals a dimer compatible with Notch1 homodimerisation in cis; N-linked glycans and interface residues are indicated; (b) Batch SAXS pair-distance distribution and Kratky plot indicate the Jagged1 EGF8-11 segment has conformational flexibility whereas the Jagged1 CRD is compact and globular. The crosshairs in the Kratky plots indicate the peak position expected for a globular protein. Figure 3: Inter- and intra-molecular interactions in the Notch1 – Jagged1 complex; (a) XL-MS analysis indicates that several sites in Jagged1 are in close proximity to the Notch1 EGF33-NRR C-terminal region; The intra- and inter-molecular interactions are confirmed in quantitative interaction experiments (SPR and MST) using targeted sites; (b) Two schematic representations indicating the architecture of the Notch1 – Jagged1 complex in a cis-setting and in a trans setting Figure 1: SAXS analysis of the full-extracellular segments of Notch1 and Jagged1 reveal conformational flexibility. Left, schematic of the 40 extracellular domains of Notch1 and the 19 extracellular domains of Jagged1. Pair-distance distribution analysis (middle column) and normalised Kratky plots (right column) of the SAXS data indicate the proteins are conformationally flexible. The crosshairs in the Kratky plots indicate the peak position expected for a globular protein.