Biophysical Evidence for Intrinsic Disorder in the C-terminal Tails of the Epidermal Growth Factor Receptor (EGFR) and HER3 Receptor Tyrosine Kinases

Abstract

Edited by Ruma BanerjeeThe epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases includes oncogenes important in the progression of breast and other cancers, and they are targets for many drug development strategies. Each member of the ErbB family possesses a unique, structurally uncharacterized C-terminal tail that plays an important role in autophosphorylation and signal propagation. To determine whether these C-terminal tails are intrinsically disordered regions, we conducted a battery of biophysical experiments on the EGFR and HER3 tails. Using hydrogen/deuterium exchange mass spectrometry, we measured the conformational dynamics of intracellular half constructs and compared the tails with the ordered kinase domains. The C-terminal tails demonstrate more rapid deuterium exchange behavior when compared with the kinase domains. Next, we expressed and purified EGFR and HER3 tail-only constructs. Results from circular dichroism spectroscopy, size exclusion chromatography with multiangle light scattering, dynamic light scattering, analytical ultracentrifugation, and small angle X-ray scattering each provide evidence that the EGFR and HER3 C-terminal tails are intrinsically disordered with extended, non-globular structure in solution. The intrinsic disorder and extended conformation of these tails may be important for their function by increasing the capture radius and reducing the thermodynamic barriers for binding of downstream signaling proteins. The epidermal growth factor receptor (EGFR)3 /ErbB family of receptor tyrosine kinases (RTKs) contains four member proteins: EGFR/ErbB1, HER2/ErbB2/neu, HER3/ErbB3, and HER4/ErbB4. These RTKs carry out important signaling functions via the sequential process of ligand binding by the extracellular domain, homo-or heterodimerization, activation of their intracellular kinase domain, and recruitment of downstream signaling proteins. These RTKs are also important oncogenic drivers in many breast, lung, and other human cancers (1). Several structural biology studies on the ErbB family have been published, and this has helped advance drug development for HER2-positive breast cancer (2). Protein crystallography studies published in 2004 showed the structure of pertuzumab bound to the extracellular domain of HER2 and lapatinib bound to the kinase domain of EGFR (3, 4). Since that time, growing structural biology-based understanding of how EGFR, HER2, and HER3 function at the atomic level has dramatically reshaped our understanding of RTKs (1, 2). Despite these advances, there is a domain in each EGFR/ErbB family protein for which little structural biology information is available; this domain is the C-terminal tail (CTT) domain. The CTTs contain numerous autophosphorylation sites that are essential for recruiting downstream signaling proteins and initiating intracellular signaling (5, 6). The CTT can also contribute to autoinhibition of the kinase domain of RTK (7). The lack of available crystallographic information on the CTT region of EGFR/ErbB fami...

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