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.

当表皮生长因子受体（EGFR）发生配体依赖性二聚化时，其中一个受体单体的内在蛋白酪氨酸激酶（PTK）活性被激活，二聚化受体可在两个羧基末端（CT）结构域内的任意八个候选磷酸化位点（P位点）处发生自身磷酸化。尽管细胞外配体结合域与细胞内PTK结构域的结构已被解析，但长度约225个氨基酸的CT结构域的结构尚未明确，推测其原因为该结构域存在无序性。CT结构域P位点的受体磷酸化在信号转导过程中至关重要，因为特定的信号效应分子会与单个磷酸化的P位点相结合。为了探究传统底物识别与CT结构域自身磷酸化反应中涉及的独特拓扑因素共同作用，如何决定P位点磷酸化的选择性，我们针对EGFR自身磷酸化事件之前的P位点/催化位点结合反应开展了粗粒度分子模拟。我们的研究结果表明，尽管普遍认为二聚化EGFR的自身磷酸化以反式（trans）方式发生，但它同样可能以相近效率通过顺式（cis）方式进行，此时两个受体单体的P位点均能达到相似的磷酸化程度。唯有与激酶最邻近的P位点-992属于例外，其与催化位点的结合仅能通过分子内反应以顺式方式发生。我们发现，P位点-992与催化位点的顺式相互作用，得益于PTK结构域N端与C端叶之间的裂隙，该裂隙使得将P位点-992连接至PTK核心的短CT结构域序列能够抵达催化位点。本研究为EGFR自身磷酸化反应提供了多项新的机制见解，并证明了粗粒度分子模拟方法在探究诸如EGFR（HER/ErbB）家族受体以及一般生长因子受体PTK的自身磷酸化复杂性方面的应用潜力。