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Published in final edited form as: J Phys Chem Lett. 2020 Nov 12;11(23):10037–10044. doi: 10.1021/acs.jpclett.0c02327

Phosphorylation-dependent Conformations of the Disordered Carboxyl-terminus Domain in the Epidermal Growth Factor Receptor

Raju Regmi †,#, Shwetha Srinivasan †,#, Andrew P Latham †, Vandna Kukshal ‡, Weidong Cui ¶,∥, Bin Zhang †, Ron Bose ‡, Gabriela S Schlau-Cohen †
PMCID: PMC8063277  NIHMSID: NIHMS1689760  PMID: 33179922

Abstract

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, regulates basic cellular functions and is a major target for anti-cancer therapeutics. The carboxyl-terminus domain is a disordered region of EGFR that contains the tyrosine residues, which undergo autophosphorylation followed by docking of signaling proteins. Local phosphorylation-dependent secondary structure has been identified and is thought to be associated with the signaling cascade. Deciphering and distinguishing the overall conformations, however, has been challenging due to the disordered nature of the carboxyl-terminus domain and resultant lack of well-defined three-dimensional structure for most of the domain. We investigated the overall conformational states of the isolated EGFR carboxyl-terminus domain using single-molecule Förster resonance energy transfer and coarse-grained simulations. Our results suggest that electrostatic interactions between charged residues emerge within the disordered domain upon phosphorylation, producing a loop-like conformation. This conformation may enable binding of downstream signaling proteins, and potentially reflect a general mechanism in which electrostatics transiently generate functional architectures in disordered regions of a well-folded protein.

Keywords: EGFR, phosphorylation, single-molecule FRET, signal transduction

Graphical Abstract

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Epidermal growth factor receptor (EGFR) is a mammalian single-pass membrane protein in the receptor tyrosine kinase family that regulates multiple cellular processes such as growth and apoptosis.1,2 Genetic mutations and over-expression of EGFR have been implicated in neurodegenerative diseases and cancers.3–7 Cancer therapeutics that bind EGFR are widely used, yet are currently limited by acquired resistance in the targeted domains.8,9 Activation of intracellular signaling pathways begins with extracellular ligand binding to EGFR, which is followed by a series of conformational changes that drive dimerization and propagate signals across the plasma membrane.10–13 This complex series of events leads to kinase activation and subsequent phosphorylation of tyrosine residues on the disordered carboxyl-terminus domain of EGFR (also referred as the C-terminal tail).14–17 The carboxyl-terminus domain is responsible for phosphorylation-dependent binding of downstream signaling proteins,18,19 as well as auto-inhibitory regulation via interaction with the kinase domain.20–24 The entire carboxyl-terminus domain is highly conserved among EGFR in all vertebrates, which is unusual in regions lacking secondary structure and indicates a critical functional role.24 While transient, partial secondary structure has been observed in local regions, the overall conformations of the carboxyl-terminus domain throughout the signaling cascade have been challenging to determine, primarily because of its disordered nature.

A large number of eukaryotic proteins are intrinsically disordered.25 Regions of 83% of the human kinases are disordered26 and nearly 50% of transmembrane proteins have at least one disordered region.27 Disordered regions have a high inclination to undergo post translational modifications, including phosphorylation, and exhibit structural flexibility to facilitate interactions with multiple binding partners.28–30 The carboxyl-terminus domain of the EGFR family (Figure 1a,b; Table S1) is a well-known example of an intrinsically disordered region that interacts with many different proteins to regulate a host of signaling pathways.27,31–33 Although an overall disordered nature of the carboxyl-terminus domain of EGFR has been established in all phosphorylation states,34,35 partial secondary structure has been observed during interaction with the kinase domain that is disrupted upon phosphorylation.34–39 Both structural and spectroscopic measurements identified local regions that exhibited signatures of this secondary structure.21,35,36,38 These results suggest that structure formation within the carboxyl-terminus domain plays a role in the signaling cascade.

Figure 1: Human epidermal growth factor receptor (EGFR) and disorder prediction for the carboxyl-terminus domain.

Figure 1:

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(a) EGFR is a membrane receptor protein which has 618-amino acid extracellular domain, and 541-amino acid intracellular domain connected by a 27-amino acid transmembrane domain. The intracellular domain consists of juxtamembrane domain, kinase domain, and a 226 (961–1186) amino acid disordered carboxyl-terminus domain. (b) Dephosphorylated (Y) and tyrosine phosphorylated (pY) constructs designed with two inter-dye separation: 154 and 66 amino acids. (c) Disorder prediction score between 0 (well-folded) and 1 (fully disordered), corresponding to the probability of given residue being part of disordered region, for each residue of the carboxyl-terminus domain.81 A score above 0.5 is predicted to be disordered. Accession numbers: 1NQL, 2JWA and 1M17.

While the local secondary structure formation has been investigated, the primarily disordered nature of the carboxyl-terminus domain means that overall structural changes have been difficult to observe. The structural heterogeneity and frequent order-to-disorder transitions of disordered proteins has limited the efficacy of many biophysical methods. Although challenging to study, it is these characteristics that are critical to their biological function. Ensemble structural and biochemical techniques have been powerful tools to investigate the organization and function of proteins, yet single-molecule Förster resonance energy transfer (smFRET) measurements have emerged as an alternative method that reveals transient structure formation hidden in ensemble measurements, including for intrinsically disordered proteins.40–47 Previous smFRET measurements identified local regions of the carboxyl terminus domain that contained partial secondary structure, which was disrupted by phosphorylation.35 A wide distribution of conformational states for the intracellular region was also found upon ATP binding,48 although it remains unclear whether these various conformations arise from differences in phosphorylation states or inherent conformational heterogeneity.20,49 Understanding the functional features, if any, in the overall conformations of the carboxyl-terminus domain and their dependence on phosphorylation is necessary to establish a mechanistic model of signal transduction in EGFR.

In this work, we investigated the influence of tyrosine phosphorylation on the overall conformation of the carboxyl-terminus domain of EGFR. We produced fluorescently-labeled isolated carboxyl-terminus domain constructs that we characterized using smFRET measurements and coarse-grained simulations. We observed an overall conformational compaction upon phosphorylation, which simulations suggest is due to contacts between charged residues inducing a loop-like conformation. While electrostatic-driven conformational changes were observed previously in intrinsically disordered proteins,50,51 the conformation identified here demonstrates the same effect within disordered regions of a well-folded protein, potentially to generate a functional architecture such as a docking site for downstream signaling proteins. In contrast to the local secondary structure previously identified, the conformational loop observed here retains its disordered nature, potentially to facilitate interactions with diverse binding partners. The identification of a phosphorylation-dependent conformational change is a step towards decoding the complex regulatory mechanisms of EGFR and other receptor tyrosine kinases, which contain similar disordered regions.2,24,52,53

We constructed dephosphorylated and tyrosine phosphorylated (pY1173) versions of the carboxyl-terminus domain with donor and acceptor dyes attached at two inter-dye separations, 66 amino acids (aa) and 154 aa, which report on local and overall conformations, respectively (Figure 1b,c, fig. S1). We produced recombinant semisynthetic phosphoproteins by using a sortase tag recognition sequence to combine donor-labeled proteins and acceptor-labeled peptides with Y1173 either phosphorylated or dephosphorylated.54,55 The modification sites were selected to minimize the number of alterations to the sequence of the EGFR protein construct. Figure 2a shows coomassie-stained and fluorescence gel images for the constructs of the full carboxyl-terminus domain. The presence of a band at ~30 kDa, the weight of the carboxyl-terminus domain,33 indicated successful recombination of the two segments (see Methods; fig. S2–S5).

Figure 2: EGFR carboxyl-terminus domain sample characterization.

Figure 2:

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(a) Protein purity before and after sortase incorporation of atto 647N labeled peptide. C1, construct with 154 aa inter-dye separation; C2, construct with 66 aa inter-dye separation. (b) Ensemble fluorescence emission spectra (λexc = 590 nm) for 154 aa (solid line) and 66 aa (dotted line) carboxyl-terminus domain constructs. The donor-only emission spectrum is shown alongside (gray). (c-d) Competition binding curves of carboxyl-terminus domain to PLCγ1. Donor fluorescence intensity from the labeled carboxyl-terminus domain in the (c) flow through and (d) elution as a function of titrated concentration of unlabeled carboxyl-terminus peptide (154 aa inter-dye separation shown here, 66 aa inter-dye separation shown in fig. S7).

Figure 2b shows ensemble fluorescence emission spectra of both unphosphorylated constructs. Emission from both donor and acceptor dyes was observed due to FRET. Using the maximum of the emission peak for the donor (625 nm) in the presence and absence of acceptor, we estimated a FRET efficiency of 24% (9 nm inter-dye distance) and 69% (6.5 nm inter-dye distance) for the 154 aa and 66 aa separations, respectively.56 The presence of FRET further confirms recombination as well as the attachment of the fluorescent dyes to the carboxyl-terminus domain. We demonstrated negligible influence of the fluorescent dyes by testing for successful binding of the phosphorylated constructs to the downstream signaling protein, PLC γ1, using a GST pulldown fluorescent intensity assay (Figure 2c,d; fig. S6–S7). The binding of the labeled phosphorylated carboxyl-terminus domain to the recognition domain SH2 of PLC γ1 was estimated by a fluorescence competition assay (see fig. S6). The similar binding affinities of the carboxyl-terminus domain and unlabeled phosphorylated peptide confirmed the functionality of the constructs after attachment of the fluorescent dyes.

Single-molecule FRET measurements were performed on immobilized constructs using a confocal microscope with time-correlated single photon counting for fluorescence lifetime measurements (Figure 3a).48 Figure 3b shows a representative single-molecule fluorescence time trace (green, donor channel; red, acceptor channel), illustrating the anti-correlated emission levels expected in the presence of FRET. Fluorescence decay curves were constructed from intensity levels in the donor channel as shown for the FRET level and for a representative level from a donor-only experiment in Figure 3c. The donor fluorescence lifetime can be used to quantify energy transfer efficiency (i.e., FRET efficiency) between donor and acceptor dyes.57,58 The FRET efficiency can be converted to distance (see Methods). Using the FRET efficiency from donor dye (atto 594, at two different positions) to acceptor (atto 647N, fixed at the end of construct) we mapped the conformations induced by tyrosine phosphorylation at Y1173 within the EGFR carboxyl-terminus domain.

Figure 3: Single-molecule FRET probes conformations of EGFR carboxyl-terminus domain.

Figure 3:

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(a) Schematic of multi-parametric single-molecule confocal scanning microscope. A 550 nm excitation source is focused with an oil immersion objective lens to scan Ni-NTA coated coverslip with immobilized carboxyl-terminus domain constructs. The emitted fluorescence is separated with an emission dichroic for separate collection of the donor (D) and acceptor (A) emission. (b) A representative single-molecule fluorescence intensity trace of a carboxyl-terminus domain construct (66 aa inter-dye separation in this case) where anti-correlated emission shows the presence of energy transfer from donor (green) to acceptor (red). (c) The donor intensity levels from (b) are used to determine the fluorescence decay dynamics. Instrument response function (IRF) is shown in gray.

Figure 4(a–d) shows donor lifetime histograms constructed from the FRET levels for the dephosphorylated (top) and phosphorylated (bottom) EGFR carboxyl-terminus domain constructs (left, 154 aa inter-dye separation; right, 66 aa inter-dye separation). The distance is quantified on the upper x-axis using a separately-characterized donor-only construct as a reference (fig. S8). The donor lifetime distribution for the dephosphorylated constructs with 154 aa inter-dye separation peaks at ~2.5 ns, corresponding to an inter-dye distance of 10.5 nm (Figure 4a). The distribution for the phosphorylated constructs with 154 aa inter-dye separation peaks at ~2 ns, corresponding to an inter-dye distance of 8.5 nm (Figure 4b). The distance change suggests that a conformational compaction occurs upon phosphorylation. In contrast to the constructs with 154 aa inter-dye separation, the donor lifetime distribution for the dephosphorylated constructs with 66 aa inter-dye separation peaks at ~2 ns, corresponding to an inter-dye distance of 8.6 nm. The distribution for the phosphorylated constructs at 66 aa inter-dye separation peaks just above 2 ns, corresponding to an inter-dye distance of 9.4 nm (Figure 4c,d). The small expansion is likely due to phosphorylation-induced loss of local secondary structure, as observed previously.35

Figure 4: Conformational states of EGFR carboxyl-terminus domain.

Figure 4:

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Histograms of smFRET measurements for dephosphorylated (a,c) and phosphorylated (b,d) versions of the constructs with 154 aa and 66 aa inter-dye separations, respectively. (e-f) Distance probabilities between the labeled residues for the 154 aa (e) and the 66 aa (f) inter-dye separations from MOFF-IDP simulations of the carboxyl-terminus domain. (g) Most common structures determined by RMSD clustering for dephosphorylated (left) and phosphorylated (right) constructs with (p)Y1173 (orange) and K1075 (blue) and labeled residues indicated with stars (green, donor; red, acceptor). Box indicates electrostatic contact between pY1173 and K1075 induced by phosphorylation. Distance between the labeled residues is shown for the 154 aa inter-dye separation.

To gain molecular insight into the conformations of the carboxyl-terminus domain, we performed coarse-grained molecular dynamics simulations of both the dephosphorylated and phosphorylated constructs. In previous work, similar coarse grained and implicit solvent models have been able to describe intrinsically disordered proteins.59–63 In this study, our coarse-grained methodology describes the carboxyl-terminus domain with alpha-carbon resolution by combining the maximum entropy optimized force field for intrinsically disordered proteins (MOFF-IDP) with secondary structure information from structure based modeling.64,65 We also include the contribution of local phosphorylation-dependent secondary structure.21,36,38 Circular dichroism measurements were performed on all constructs. Consistent with previous work investigating other phosphorylation sites,35 the measurements showed a 20% increase in unordered content after phosphorylation (fig. S9), which was incorporated into the model (see Methods for details). To confirm that our conformational ensembles agree with previous experiments, we first computed the radius of gyration (Rg) for both the dephosphorylated and phosphorylated carboxyl-terminus domains (fig. S10, Table S2). The mean Rg of the dephosphorylated protein (4.66 nm) agrees with previous experimental observations (4.66 nm).33 The mean Rg of the phosphorylated protein (4.39 nm) is smaller than in the dephosphorylated case, consistent with the reduced distance for the 154 aa inter-dye separation observed by smFRET.

For a more direct comparison with the smFRET results, we calculated the distributions of distances between the alpha-carbons of the labeled residues. All experimental and simulated distances are summarized in Table S2. For the 154 aa separation (Figure 4e), the peak of the distribution is at 9.2 nm for the dephosphorylated construct and decreases to 7.6 nm for the phosphorylated version. The distances extracted from the experimental measurements include the contribution of the dyes and their linkers, and so are ~2 nm longer than the simulated distances (fig. S11).66 For the 66 aa separation (Figure 4f), the peak of the distribution is at 5.1 nm for the dephosphorylated construct and increases slightly to 5.4 nm for the phosphorylated version due to loss of local secondary structure.35 While the model underestimates the distances for the 66 aa separation, the good agreement for the 154 aa and the mean Rg suggest that the model captures the overall conformations, which are the focus of this work.

Examination of the simulations reveals that long range electrostatic interactions drive large scale changes in the protein structure upon phosphorylation. The negatively-charged phosphorylated tyrosine (pY1173) forms a stable electrostatic contact with two neighboring positively-charged residues (K1075 and R1076; fig. S12). This contact is populated 50% of the time in the phosphorylated protein, as opposed to 0.1% of the time in the dephosphorylated case. In explicit solvent simulations using the MARTINI force field and umbrella sampling,67–69 a free-energy minimum is observed at a 1.5 nm distance between K1075 and pY1173 alpha carbons for the phosphorylated construct; the residues remained in spatial proximity over an independent, unbiased 2 μs long simulation (fig. S13). The free-energy minimum and the spatial proximity disappeared for the unphosphorylated construct (fig. S13). Finally, in fully atomistic simulations conducted on the loop region of the phosphorylated protein, this contact remains stable (fig. S14).70 Although there are 20 positively charged residues within the domain, these are the only two neighboring ones, which may play a role in the stable nature of the interaction (fig. S1, Table S1).

To investigate the overall structure that results from the electrostatic contact, we performed RMSD clustering on the ensembles from the coarse grained simulations. The clusters show that the region between K1075 and pY1173 is significantly more compact after phosphorylation due to the electrostatic contact (fig. S15). The most representative structure by RMSD clustering shows that the stable electrostatic contact promotes the formation of a loop-like structure, while its absence for the dephosphorylated tyrosine leads to a more extended configuration (Figure 4g). For a 154 aa separation, the loop formation brings the residue with the donor label (D1) closer to the one with the acceptor whereas, for a 66 aa separation, both the labeled residues lie on one side of the loop, and so do not move closer together upon its formation. The loop formation is robust to the presence or absence of local secondary structure, as it is observed even in simulations where the phosphorylation-dependent secondary structure is not included (fig. S16). This further supports the conclusion that the loop is distinct from the previously identified loss of local secondary structure.

Our results show that an overall conformational change occurs in the carboxyl-terminus domain and is controlled by electrostatic interactions. Phosphorylation introduces electrostatic interactions that induce the conformational change, similar to observations in other proteins. For example, phosphorylation-dependent electrostatic interactions were shown to promote local conformational changes in human kinase proteins.71 In addition, in the enzyme isocitrate dehydrogenase, mutants that replaced the phosphorylation site with a negatively charged residue mimicked the phosphorylated version.72 The similarity between negatively charged residues and phosphorylation sites is even thought to have played an important role in the evolution of phosphorylation sites.73 Electrostatic interactions with phosphorylated residues are likely to be particularly important in disordered regions, which contain a relatively large number of charged residues that interfere with formation of the secondary structures.74 Electrostatic interactions have been shown to drive the formation of structured regions for a intrinsically disordered protein.44,50,51 Here, we showed that they can have the same effect for a disordered region within a well-folded protein.

Electrostatic-driven loop formation in the EGFR carboxyl-terminus domain may trigger binding of the downstream signaling proteins.26,75 Intrinsically disordered regions are known to adopt heterogeneous conformations, and it is this multiplicity of conformations that facilitates binding, including with multiple interaction partners. Multiple conformations also serve to reduce thermodynamic barriers for binding reactions and to increase capture radii for enhanced kinetics.76 In the case of receptor tyrosine kinases, disordered regions such as the carboxyl-terminus domain play a role both in the regulation of kinase activity and also in the formation of complexes with seven known signaling proteins.31,77 Previous studies suggest that the carboxyl-terminus domain of EGFR has local secondary structure during kinase interaction, which is disrupted, along with the interaction, upon phosphorylation.21,36,38 This has been shown to lead to a local expansion,35 as seen in our measurements as well. Our additional observation of an overall compacted conformation of the domain suggests that phosphorylation also leads to a transition into a loop structure formed from the disordered region. Thus, while previous work identified a local order-to-disorder transition upon phosphorylation, here we complement and expand upon this work by reporting signatures of a disorder-to-disorder transition that spans the domain. This transition had been hidden in many ensemble measurements due to the challenges associated with characterizing disordered proteins. The loop structure could interact with the domains that recognize and bind phosphorylated tyrosines, the phosphotyrosine binding domain and Src homology 2 domain.78 The two distinct structural motifs identified in the carboxyl-terminus domain, secondary structure and conformational loop, would then carry out its two roles in signal transduction. Thus, the transition to a loop structure may be the conformational switch that activates binding of proteins while retaining the disordered character that enables the formation of diverse signaling complexes.

In this work, we report the observation of a conformational compaction upon phosphorylation in the carboxyl-terminus domain of EGFR. Based on our experimental and computational results, we assign the conformational change to a disorder-to-disorder transition that forms a loop through electrostatic interactions between the phosphorylation site and charged residues. While electrostatic-driven structure formation was previously observed in intrinsically disordered proteins,50,51 we observe this phenomenon within a disordered region of a well-folded protein. The loop conformation identified here is an alternative structure that could serve to recruit signaling proteins for efficient signal transduction, in contrast to the secondary structure formation implicated in kinase interaction.52 EGFR is a successful target for cancer therapeutics, yet resistance has emerged to current binding sites, which introduces a role for alternative targets.8,9,79 The carboxyl-terminus domain is conserved across all vertebrates and contains key tyrosine residues for signaling.24 Sites within and conformations of the carboxyl-terminus domain could thereby provide alternatives for inhibiting EGFR-induced signaling as a cancer treatment, consistent with other therapeutics that target disordered proteins.80 Moreover, building a mechanistic understanding of EGFR signal transduction has the potential to guide the development of the next-generation therapeutics.

Supplementary Material

Supplementary Material

Acknowledgement

This work was supported by the NIH Director’s New Innovator Award 1DP2GM128200-01 and a Beckman Young investigator Award (to G.S.S.-C.). A.L. and B.Z. were supported by the National Institutes of Health Grant 1R35GM133580-01 and A.L. further acknowledges support by the National Science Foundation Graduate Research Fellowship Program. This work was also supported by National Institutes of Health Grants R01 CA161001 (to R. B.) and 8P41 GM103422. We thank Michael Gross and the National Institutes of Health/National Center for Research Resources Mass Spectrometry Resource for access to MS instrumentation and acknowledge John Monsey for the help with sample preparation. We also thank Xingcheng Lin for help with simulations using the MARTINI force field. G.S.S.-C. also acknowledges a Sloan Research Fellowship in Chemistry, a Smith Family Award for Excellence in Biomedical Research, and a CIFAR Global Scholar Award.

Footnotes

Supporting Information Available

Materials and methods, amino acid sequence, triton X-100 titration, trypsin treatment, calf intestinal phosphatase treatment, mass spectroscopy results, flow chart of Sh2-binding reaction, Sh2-binding reaction with 66 aa constructs, donor-only lifetime histograms, circular dichroism spectroscopy, MOFF-IDP radius of gyration, dyes and their accessible volumes, MOFF-IDP contact frequency map, free energy of loop formation with MARTINI modeling, CHARMM-36m frequency of key contacts, structural clustering results for MOFF-IDP simulations, MOFF results without changes to secondary structure, table for amino acid composition, table for distance comparison. This information is available free of charge via the Internet at http://pubs.acs.org.

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