Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR (HER/ErbB) family receptors and growth factor receptor PTKs in general.Author Summary: The epidermal growth factor receptor (EGFR) is one of a large group of cell surface receptors that allow cells to respond to growth-stimulating signals in their environment. Upon sensing of growth factor, the EGFR is activated, which triggers a signaling cascade leading to the cell nucleus and ultimately initiating cell division. The first event following receptor activation is an intramolecular kinase reaction that results in the introduction of phosphate groups onto several specific amino acids (phosphorylation sites or P-sites) in the tail of the EGFR protein. Thus, the tail of the receptor undergoes self-phosphorylation, which involves conformational motions enabling the various P-sites to access the catalytic site. The structure of the tail of the receptor is unknown, and hence the mechanism of the self-phosphorylation reaction is not well understood. To investigate this mechanism, we generated a structural model of the EGFR protein and performed computer simulations of EGFR P-site/catalytic site binding reactions. These simulations indicated how the distribution of P-sites along the tail of the receptor and restrictions in molecular movements of the tail lead to selectivity in the phosphorylation of the different P-sites. Our simulations yielded unique insights into the mechanism of EGFR self-phosphorylation that have important biological implications.