Periodic Reporting for period 1 - EGFR-Activ (Epidermal growth factor receptor (EGFR) activation by cytohesins - Structural Insights)
Reporting period: 2015-05-04 to 2017-05-03
The overall objectives of the projects focused on a better understanding of cytoplasmic components that may play a, yet not well characterized, role in EGFR activation. Better insights into these factors may help to develop new therapeutic strategies to target various EGFR-related pathogenic signalling pathways. To obtain reliable information of these molecular details, it is of fundamental importance to also apply high-resolution approaches that on the one hand provide structural information in a site-resolved manner and on the other hand are applicable to environmental conditions that sufficiently mimic native conditions. For this reason, mainly high-resolution solution-state NMR was used at temperature, buffer and environmental conditions such as detergent free membrane mimetics at physiological pH and temperature. The structural NMR studies in particular focused on the role of its juxtamembrane domain (JM), the membrane environment and cytoplasmic co-factors (i.e. the Sec7 of cytohesin-2). Overall, we succeeded in identifying interactions and characterizing structural details of the JM-Sec7 binding as well as JM-membrane association. For the latter, we also identified a dependence of the lipid composition of the membrane mimetic.
The obtained data may be transferred towards academic and non-academic environments and contribute to the global knowledge of EGFR-signalling and to the discovery of novel therapeutic agents with the potential to save human lives.
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.
It can be anticipated that the cytoplasmic factors, including the ones that were characterized in this project, will play an increasingly important role in the development of an urgently needed new generation of therapeutic strategies targeting the EGFR signalling pathway.