EGFR and HER 3 C-terminal tails are intrinsically disordered Biophysical Evidence for Intrinsic Disorder in the C-terminal Tails of the EGFR and HER 3 Receptor Tyrosine Kinases

The 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 Cterminal tail that plays an important role in autophosphorylation and signal propagation. To determine if 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 to the ordered kinase domains. The C-terminal tails demonstrate more rapid deuterium exchange behavior when compared to the kinase domains. Next, we expressed and purified EGFR and HER3 tail-only constructs. Results from circular dichroism spectroscopy, size-exclusion chromatography with multi-angle light scattering, dynamic light scattering, analytical ultracentrifugation, and small angle Xray 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. INTRODUCTION The Epidermal Growth Factor Receptor (EGFR/ErbB) family of receptor tyrosine kinases (RTK’s) contains four member proteins: EGFR/ErbB1, HER2/ErbB2/neu, HER3/ErbB3, and HER4/ErbB4. These RTK’s carry out important signaling functions via the sequential process of ligand-binding by the extracellular domain, homoor heterodimerization, activation of their intracellular kinase domain, and recruitment of downstream signaling proteins. These RTK’s 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, 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M116.747485 The latest version is at JBC Papers in Press. Published on November 21, 2016 as Manuscript M116.747485 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 5, 2016 hp://w w w .jb.org/ D ow nladed from EGFR and HER3 C-terminal tails are intrinsically disordered HER2, and HER3 functions at the atomic level have dramatically re-shaped our understanding of RTK’s (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 RTK’s kinase domain (7). The lack of available crystallographic information on the CTT region of EGFR/ErbB family RTK’s led us to examine if these proteins lack a stable secondary and/or tertiary structure. Intrinsically disordered regions (IDRs) represent an emerging area of interest in medicine. IDRs are regions within proteins that exhibit high flexibility and may lack a secondary or tertiary structure, but are still able to carry out important biological functions (8-15). Algorithm prediction methods indicate that around 25-30% of eukaryotic proteins can be categorized as having disordered regions (16). These disordered regions provide certain advantages in protein-protein interactions including a larger hydrodynamic radius (17,18), faster onand off-rates of binding (19), high binding specificity (20), and the ability to adopt different conformations depending on the binding partner (9,21). Correlation studies have revealed a high propensity for disordered regions to undergo post-translational modification, particularly phosphorylation (22). Because traditional methods, such as NMR and X-ray crystallography, were developed to study stable protein structures, IDRs have been more difficult to analyze due to difficulties in obtaining high concentrations or representative protein crystals (11,23). Correlation studies suggest a strong association between IDRs and human cancerassociated proteins (24) and, therefore, identifying and analyzing IDRs in cancer-related proteins is vital in understanding how they function. Protein kinases demonstrate a high degree of specificity in facilitating phosphorylation, yet many are able to perform such interactions with multiple substrate partners (25). An analysis of the human kinome shows that as many as 83% of kinase genes contain IDRs, which could facilitate these multiple interactions; RTK’s being involved in more protein-protein interactions than any other kinase group, thus potentially pointing to the involvement of IDRs in their recognition mechanisms (26). Multi-sequence alignment (27) of EGFR/ErbB family RTK’s shows that the kinase domain sequences are highly conserved between all four family members. However, the CTT regions are highly divergent between each EGFR/ErbB family member. One previous study using circular dichroism (CD) spectroscopy indicated that the EGFR CTT is rich in α-helical and β-sheet content (6), but a later study used coarse-grained modeling to show many possible conformations of the EGFR CTT based on the assumption that it is naturally disordered (28). A high degree of flexibility in the CTT would provide distinct advantages to EGFR interaction with downstream Src homology-2 (SH2) and phosphotyrosine-binding (PTB) domains during signaling functions (6). In this work, we examine the biophysical properties of the CTT of EGFR and HER3. The motivation of this study is to gather structural information on the CTT region to determine if the tails are highly dynamic, disordered regions. First, we measured the conformational dynamics of intracellular half (ICH) constructs of EGFR/ErbB family members using amide hydrogen/deuterium exchange mass spectrometry (HDX-MS). This information is used to compare the CTTs to the kinase domains. We also expressed and purified EGFR and HER3 CTT only constructs and demonstrated that they are functional because they are recognized and phosphorylated by EGFR family kinases and once phosphorylated, can be bound by the Grb2 SH2 domain. Using these CTT constructs, we performed multiple biophysical analyses, including CD spectroscopy, sizeexclusion chromatography with multi-angle light scattering (SEC-MALS), dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and small angle X-ray scattering (SAXS). The results of these methods support the hypothesis that the EGFR and HER3 CTTs are IDRs with extended, non-globular structure in solution. RESULTS Disorder Predictions on EGFR, HER2 and HER3 We used computation algorithms to provide an initial survey of disordered regions in the EGFR/ErbB family of RTK’s. Predictor of 2 by gest on N ovem er 5, 2016 hp://w w w .jb.org/ D ow nladed from EGFR and HER3 C-terminal tails are intrinsically disordered Natural Disordered Regions (PONDR) is a collection of algorithms that use an amino acid sequence to predict native disorder (29,30). The VL-XT algorithm assigns a disorder propensity score for disorder on a residue by residue basis (29-31). The VL-XT prediction for EGFR, HER2 and HER3 can be seen in figure 1. PONDR scores greater than 0.5 indicate predicted disorder and scores less than 0.5 indicate predicted order. Kinase domains of the three EGFR/ErbB family members show mostly predicted order, whereas the CTT in each show large regions of predicted disorder. The amino acid composition of the CTT and kinase domains of both EGFR and HER3 is shown in table 1. When comparing the CTTs to the kinase domains in both proteins, we observe that the CTTs are more enriched in polar, uncharged residues and prolines, while relatively depleted in hydrophobic residues. For EGFR, polar, uncharged residues make up 30.1% of the CTT versus 14.9% of the kinase domain. Prolines make up 10.6% of the EGFR CTT versus 5.4% in the kinase domain and hydrophobic residues (excluding tyrosine which can become phosphorylated) are 27.4% of the EGFR CTT versus 38.9% of the kinase domain (Table 1). Similar values are seen with HER3 CTT (Table 1), despite its primary sequence divergence from EGFR CTT (22% sequence identity between EGFR and HER3 CTT, calculated with Clustal Omega). The lowered hydrophobicity provides a simple explanation as to why the tails would not form a hydrophobic core and therefore be disordered in an aqueous environment. These predictions are a useful tool for gaining a general view of disordered regions in a protein sequence, but direct empirical evidence would better support the hypothesis of the CTTs as being IDRs. Hydrogen/Deuterium Exchange Mass Spectrometry of EGFR/ErbB Family RTK’s HDX-MS is a highly useful technique to identify and study IDRs (32-36). HDX-MS provides kinetic information about the exchange rate of amide hydrogen atoms along the backbone of a protein for solvent deuterons. This rate of H/D exchange is affected by factors such as the presence of strong hydrogen bonds, secondary structure, and tertiary structure (37-40). IDRs show very rapid H/D exchange rates, on the order of milliseconds to seconds (32-36). HDX-MS provides local information about different regions of the protein and readily distinguishes folded, globular regions from IDRs. We performed HDXMS experiments by exposing the protein to deuterium oxide (D2O) over two ranges of time points, 108 m