Automated studies of the structure of multimer complexes of the EGFR on cells at ~4 nm resolution: Elucidating structure-function relationships in cancer
- Abstract number
- 76
- Presentation Form
- Submitted Talk
- Corresponding Email
- [email protected]
- Session
- Stream 6 (Frontiers): Development and Applications in Super Resolution Microscopy
- Authors
- Dr Sumanth Iyer (1), Dr Benjamin Davis (1), Dr Laura Zanetti Domingues (1), Dr Selene Roberts (1), Dr Daniel Rolfe (1), Professor Marisa Martin-Fernandez (1)
- Affiliations
-
1. Central Laser Facility, STFC Rutherford Appleton Laboratory
- Keywords
EGFR, Tyrosine Kinases, single-molecule, fluorescence, cancer, signalling, super-resolution
- Abstract text
A crucial step in the evolution of multi-cellular organisms was the emergence of receptor tyrosine kinases (RTKs). Trafficked to the plasma membrane, their role is to transduce signals in response to cognate growth factor ligands. Epidermal growth factor receptor (EGFR) was the first RTK to be cloned and this receptor is of paramount importance to cell function and human health as mutations in EGFR and gene amplification are observed in many human cancers. Indeed, cancer is the second cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018. Despite ~4 decades of EGFR research, the information on EGFR structure derived from cell-free methods still does not explain the mechanisms underpinning either its normal function, or its dysregulation in cancer. Determining the structure-function relationships of EGFR in cells is of paramount important to find better cancer treatments.
As part of efforts to determine the structure of the EGFR in the physiological cell context, we developed a single molecule localisation imaging-based, super-resolution method, coined fluorophore localisation imaging with photobleaching (FLImP). By exploiting molecular fluorescent photobleaching steps to localise molecules co-located within diffraction-limited spots, this method measures the lateral separation (r) between such molecules with ~4 nm resolution. Using FLImP in concert with fluorescence resonance energy transfer (FRET), to measure EGFR residue-to-plasma membrane separation (z dimension), and long-time-scale molecular dynamic simulations, we determined the atomic resolution structures of ligand-bound, and ligand-free EGFR dimers and oligomers at the plasma membrane of cells (Needham et al., Nat Comms 2016; Zanetti-Domingues et al., Nat Comms 2018). These results shed new light on the mechanisms of EGFR autoinhibition and phosphorylation, in both normal and dysregulated signalling.
We have since added a second spatial dimension to FLImP (from lateral r to independent measurement of x,y dimensions). In addition, to make FLImP easier to use, we automated the data collection and analysis processes. Using a palette of strategic mutations, we have recently found that EGFR uses an autoinhibitory, fail-safe mechanism, which relies on extracellular, transmembrane, and intracellular contacts, some of which are resistant to the onset of cancer mutations. These data are currently beginning to unravel the structure-function relationship encoding the behaviour of cancer mutants responsible for the development of therapeutic resistance, thus revealing potential new candidates for therapeutic intervention.
- References
Needham, S.R., Roberts, S.K., Arkhipov, A., Mysore, V.P., Tynan, C.J., Zanetti-Domingues, L.C., Kim, E.T, Losasso, V., Korovesis, D., Hirsch, M., Rolfe, D.J., Clarke, D.T., Winn, M., Lajevardipour, A., Clayton, A.H.A. Pike, L.J., Perani, M., Parker, P.J., Shan, Y, Shaw, D.E. & Martin-Fernandez, M.L. EGFR oligomerization organizes kinase-active dimers into competent signalling platforms. Nat Comms (2016) DOI: 10.1038/ncomms13307
Laura C. Zanetti-Domingues, Dimitrios Korovesis, Sarah R. Needham, Christopher J. Tynan, Shiori Sagawa, Selene K. Roberts, Antonija Kuzmanic, Elena Ortiz-Zapater, Purvi Jain, Rob C. Roovers, Alireza Lajevardipour, Paul M.P. van Bergen en Henegouwen, George Santis, Andrew H.A. Clayton, David T. Clarke, Francesco L. Gervasio, Yibing Shan, David E. Shaw, Daniel J. Rolfe, Peter J. Parker, and Marisa L. Martin-Fernandez. The architecture of EGFR's basal complexes reveals autoinhibition mechanisms in dimers and oligomers. Nat Comms (2018) DOI:10.1038/s41467-018-06632-0.