Integrated Computational/Experimental Identification, Phosphorylation and Stapling of an Intramolecular Auto-Inhibitory Helical Segment C-Terminal to Breast Cancer Her2 Kinase Domain

Human epidermal growth factor receptor 2 (Her2) is a member of the ErbB family of transmembrane receptor tyrosine kinases implicated in various cellular processes and has been approved as an effective therapeutic target of invasive breast cancer. The protein contains an intracellular tyrosine kinase domain that has been widely documented over the past decades. Instead, we herein concerned the C-terminal tail region next to the kinase domain, which has received less attention but may play an important role in regulating the kinase activity. We systematically examined the intramolecular interaction between the kinase domain and the C-terminal tail of Her2 protein at the structural level and identified a short C-terminal sequence as an intramolecular auto-inhibitory helical segment (iAHS) that is connected to the kinase domain through a flexible linker and can reversibly interact its specific binding pocket nearby the active site of Her2 kinase, thus regulating the kinase activity through an auto-inhibitory approach. We demonstrated that the Tyr1005 phosphorylation can significantly destabilize the binding potency of iAHS to the kinase domain, which thus works as a molecular switch to dynamically regulate the iAHS binding and unbinding by Tyr1005 dephosphorylation and phosphorylation, respectively. In addition, it was found that chemical stapling of the iAHS-derived peptide can effectively improve the peptide affinity to the kinase domain, which constrains the free peptide into a native-like helical conformation to promote the binding. The resulting stapled counterpart can competitively displace the fluorescently labeled native iAHS peptide from the specific binding pocket of the Her2 kinase domain with high potency. Based on the above findings, we herein proposed a mechanism by which the C-terminal iAHS segment contributes to the dynamic regulation of Her2 activity through an intramolecular auto-inhibitory approach, that is, the Tyr1005 dephosphorylation promotes the binding of iAHS to its specific binding pocket nearby the active site of Her2 kinase domain, thus inhibiting the kinase activity by competitively disrupting substrate entrance to the site. Conversely, the Tyr1005 phosphorylation destabilizes the bound state of iAHS with Her2 kinase domain, thus enhancing the kinase activity.