Abstract
Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. This transmembrane signaling is generally initiated by ligand binding to the receptors in their monomeric form. While subsequent receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution of monomeric receptors has been challenging to isolate due to the complexity and ligand-dependence of these interactions. By combining membrane nanodiscs produced with cell-free expression, single-molecule Förster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes upon ligand binding are common to single-pass membrane proteins.
Introduction
Receptor tyrosine kinases, surface receptors present in all cell types across the animal kingdom, regulate major cellular functions, including cell division and survival1,2,3. The regulatory signals are primarily initiated by extracellular ligand binding to monomeric receptors, which causes intracellular autophosphorylation and subsequent recruitment of adapter proteins to the phosphorylated residues1. Epidermal growth factor receptor (EGFR), a prototypical receptor tyrosine kinase, has been extensively investigated as its aberrant expression leads to diseases such as cancer and diabetes4,5. Binding of its most studied ligand, the epidermal growth factor (EGF), induces a conformational expansion of the extracellular region, enabling dimerization of EGFR6,7. This expansion as well as other ligand-induced changes have been well characterized for the extracellular region8,9. The corresponding changes to the intracellular region, however, have only been accessible for oligomers due to the limited window between ligand binding and dimerization10. Analysis of fragmented domains has emerged as an alternative strategy that can isolate the conformations associated with signaling states11,12,13,14,15,16, yet these domains cannot be used to visualize how extracellular stimuli are propagated across the plasma membrane such as through extracellular/intracellular conformational coupling17. In 47% of membrane proteins, including EGFR, a single transmembrane helix spans the plasma membrane18. Although different conformations of this helix alone have been observed19,20,21, how, or even whether, the single helix can support extracellular/intracellular conformational coupling to mediate a signaling cascade has been largely unexplored.
Prior to ligand binding, 95% of EGFR is found in its monomeric form in cells22. While EGF-induced dimers have been long established as an active form of the receptors, emerging evidence suggests that other oligomerization states of EGFR also play a role in phosphorylation and signaling6,23,24. Both homo- and heterodimerization between members of the EGFR family has been observed25,26. The nature of the dimer can enhance ligand affinity or protein binding, providing an alternative mechanism to control signaling levels27,28,29,30. Depending on the lipid composition of the plasma membrane, ligand-induced formation of multimers induces stronger and more complete phosphorylation of the tyrosines and a wider dynamic range of EGFR responsiveness24,31,32,33. Furthermore, early studies suggested EGFR signaling can occur even in the presence of an antibody that prevented dimer and multimer formation23,34. Consistently, no homodimerization was observed for the ligand epigen, yet it still induces signaling35,36,37. Despite these multiple lines of evidence, the behavior of monomeric EGFR prior to oligomerization and its contribution, if any, to the signaling pathway have not yet been determined. Here, we use a multidisciplinary approach involving mutagenesis, single-molecule Förster resonance energy transfer (smFRET), molecular dynamics (MD) simulations, and cellular phosphorylation studies to isolate and investigate extracellular/intracellular conformational coupling within monomeric EGFR and its impact on cell signaling.
Results
Labeled EGFR monomers in nanodiscs
Cell-free expression was used to produce full-length EGFR monomers embedded in lipid bilayer nanodiscs and free of cellular interaction partners (Fig. 1a, Supplementary Figs. 1–3)38. A FRET donor dye (snap surface 594) was covalently attached to the C-terminus of the protein and an acceptor dye (Cy5) was introduced as a labeled lipid within the bilayer (Supplementary Fig. 4)39. The functional, folded conformation of the labeled receptors was implied with ATP-dependent phosphorylation for the intracellular region (Supplementary Fig. 2, Supplementary Tables 1, 2) and specificity of ligand binding for the extracellular region (Fig. 2), consistent with previously published western blot and fluorescence-based phosphorylation assays with similar preparations38,39. Intact, full-length monomeric EGFR was further purified spectroscopically by immobilizing nanodiscs on a coverslip at dilute concentration and only selecting receptors with a single donor and acceptor for analysis (Fig. 1b, Supplementary Fig. 5).
