Abstract
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.
Highlights
Classical polypeptide growth factor receptors are intrinsic membrane proteins, each possessing a ligand binding domain in the N-terminal extracellular portion of the molecule, a protein tyrosine kinase (PTK) domain in the C-terminal intracellular portion, and a single transmembrane (TM) domain in the intervening sequence [1]
There have been exciting recent applications of all-atom molecular dynamics methods in investigating various structural transitions associated with epidermal growth factor receptor (EGFR) function, the large number of atoms in our dimeric EGFR structure and the presumed long time scale of receptor CT domain self-phosphorylation events precluded their simulation by all-atom explicit solvent molecular dynamics
By separately docking each of the eight nine-amino acid phosphorylation sites (P-sites) sequences in the active site of the active conformation PTK domain structure, we identified a set of ‘‘native’’ contacts characterizing each P-site/active site interaction, with identical contacts used to characterize the active site interactions of the two copies of each P-site present in the dimeric receptor and the potential cis or trans interaction of each P-site with the active site of either the receiver molecule or the activator molecule
Summary
Classical polypeptide growth factor receptors are intrinsic membrane proteins, each possessing a ligand binding domain in the N-terminal extracellular portion of the molecule, a protein tyrosine kinase (PTK) domain in the C-terminal intracellular portion, and a single transmembrane (TM) domain in the intervening sequence [1]. The intrinsic PTK activity of the intracellular domain is activated upon a ligand-dependent dimerization of receptor monomers, with activation resulting in the self-phosphorylation (autophosphorylation) of both monomers on multiple tyrosine residues. While the differing sequences of phosphorylated receptor P-sites results in a selectively in their recruitment and activation of downstream signaling effectors, it is not known what determines the efficiency with which individual P-sites become phosphorylated upon receptor activation. This is in part because the structures of the various receptor CT domains are unknown, perhaps reflective of their generally disordered nature, and because the dynamics of these domains have not been examined
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