The performed research focused on the understanding of how membrane environment and cytoplasmic factors (cytohesins) modulate EGFR activation. For this we have used Nuclear Magnetic Resonance (NMR) to study the interaction between the Juxtamembrane (JM) segment of EGFR and the Sec7 domain of cytohesin-2 (ARNO), postulated to act as an EGFR activating factor. Furthermore, we also studied the influence of the composition of the cell membrane. The first step was to perform the backbone assignment of JM and Sec7. This was done using a standard triple resonance approach. The data for Sec7 revealed a well folded structure, composed by 10 alpha-helices, in good agreement with previously determined structures. For JM the data showed that, in solution, it’s structure is mainly random coil.
The next step involved the study of the interaction of JM with Sec7 and the identification of the residues involved in this interaction. For this we used NMR chemical shift perturbations by performing two different titrations: first, we titrated Sec7 with JM and then we titrated JM with Sec7. The data clearly showed that JM and Sec7 interact and allowed to precisely identify the residues involved in binding from both partners. Interaction of JM with Sec7 was also confirmed by using Saturation Transfer Difference NMR (STD-NMR).
JM-membrane interactions were investigated using lipid bilayer nanodiscs with defined lipid composition. Different membrane binding modes of the JM domain were identified depending on lipid charge content.
Overall our results help to clarify, so far unfamiliar and controversially discussed, mechanism of cytohesin-mediated EGFR signalling and open the door to the possible therapeutic exploitation of this mechanism.
In the process of this work, new NMR methodologies were developed that are tailored to the study of complex systems and have the potential to improve the amount and quality of the acquired data, thus facilitating the study of these systems. Additionally, the usage of detergent-free membrane mimetics for NMR-based structural studies was carefully evaluated. Large parts of the results of this project have already been communicated to the scientific community via three publications in peer-reviewed international journals (Viegas A, et al., 2016, Biological Chemistry, 397 (12), 1335-1354; Viennet T, Viegas A, et al., 2016, Ang. Chem. Int. Ed., 55, 10746 –10750; Viegas A, et al., 2016, JBNMR, 64, 9–15), two invited oral presentations (EUROMAR, 2015 – Prague, Czech Republic; Instituto de Tecnologia Química e Biológica, 2016 – Lisbon, Portugal) and two poster presentations (EUROMAR, 2015 – Prague, Czech Republic; 38th FGMR Discussion Meeting, 2016 – Düsseldorf, Germany). Additional publications, including all so far not published results, are currently in preparation.
