EGFR in different membrane environments.

(a) Full-length EGFR (gray) embedded in a nanodisc. The nanodisc is a lipid bilayer (beige) belted by an amphiphilic apolipoprotein (dark gray). EGFR consists of a 618-amino acid extracellular region that binds EGF (orange), a 27-amino acid transmembrane-spanning domain, and an intracellular region, which is a 37-amino acid juxtamembrane domain, a 273-amino acid kinase domain and a 231-amino acid disordered C-terminal tail. Green and maroon spheres indicate the donor and acceptor dyes, respectively (Supplementary Fig. 1). (b) Mean of zeta potential distributions for EGFR in nanodiscs containing increasing amounts of anionic lipids (0%, 15%, 30% and 60% POPS). Error bars are from three technical replicates. (c) Ensemble fluorescence emission spectra (λexc= 385 nm) of EGFR embedded Laurdan containing nanodiscs with increasing cholesterol.

Membrane composition influences EGFR function through ATP binding.

(a) Full-length EGFR in nanodiscs with atto647N γ ATP (red sphere) and snap surface 488 or snap surface 594 (green sphere). (b) Extent of ATP binding in different anionic environments quantified using the intensity of atto 647N (top) band normalized by the amount of EGFR produced as extracted from the intensity of ss488 band (center) as a function of negatively charged POPS lipids (bottom) in the absence (purple) and presence (orange) of EGF. Error bars from three independent biological replicates. (c) EGFR intracellular domain indicating ATP binding site-C-terminus distance and ATP-lipid contacts measured from molecular dynamics simulations. (d) Accessibility of ATP binding site quantified through the contact number between the ATP binding site and lipids in the absence (purple) and presence (orange) of EGF. Probability distributions of the distance between residue 721, the closest residue to the ATP binding site,60 and EGFR C-terminus for (e) neutral (0% POPS) and (f) 30% anionic lipids (30% POPS) without EGF (top); with 1 µM EGF (bottom). Dotted lines indicate the medians on all histograms with corresponding distances on upper x-axis. (g) Schematic of multiparametric single-molecule confocal microscope. (h) Fluorescence intensity for a representative image (λexc = 550 nm) where green spots are immobilized EGFR nanodiscs. (i) Representative fluorescence time trace from single-molecule FRET experiments showing number of detected photons for each 100 ms interval as intensity traces (green for donor; red for acceptor) with the average for each period of constant intensity (black solid line) and the corresponding donor lifetime (black dashed line). smFRET donor lifetime distributions with atto647N IATP as acceptor and snap surface 594 as donor in (j) neutral (0% POPS) and (k) 30% anionic lipids (30% POPS) without EGF (top); with 1 µM EGF (bottom).

Median distances between the ATP binding site (residue 721) and the C-terminal end of EGFR from smFRET experiments and simulations.

The distance values were extracted as the medians of the distributions in Figs. 2e, f, j, k in the main text. The numbers in parenthesis indicates the 95 % confidence interval for experiments and the minimal and maximal median value from three equal partitions of data for simulations. Ro = 7.5 nm for snap surface 594 and atto 647N

EGFR intracellular conformations depend on membrane composition of the nanodisc.

(a) Full-length EGFR in nanodiscs with partially anionic lipids (red). The negatively charged residues on the C-terminal tail are indicated in red and the positively charged residues on the kinase domain are indicated in blue. smFRET donor fluorescence lifetime distributions in (b) 100% POPC, 0% POPS; 85% POPC, 15% POPS; 70% POPC, 30% POPS; 40% POPC, 60% POPS without EGF (top); with 1 µM EGF (bottom). (c) Full-length EGFR in nanodiscs with cholesterol (teal). (d) smFRET donor fluorescence lifetime distributions in 92.5% POPC, 7.5% cholesterol; 80% POPC, 20% cholesterol without EGF (top); with 1 µM EGF (bottom). (e) Full-length EGFR in nanodiscs with cholesterol and anionic lipids. (f) smFRET donor fluorescence lifetime distributions in 62.5% POPC, 30% POPS, 7.5% cholesterol; 50% POPC, 30% POPS, 20% cholesterol without EGF (top); with 1 µM EGF (bottom). Dotted lines indicate the maxima from a global fit of all lifetime distributions to a double Gaussian distribution model using maximum likelihood estimation. The maxima correspond to a compact and an open conformation with a distance of 8 nm and 12 nm, respectively, between the EGFR C-terminal tail and the membrane bilayer. (g) The amplitude of the open conformation (in %) in all the eight different membrane compositions in the absence (purple) and presence of EGF (orange). (h) The change in amplitude induced by EGF (black). The amplitude change upon EGF addition is high (22% – 55%) in 0% – 30% POPS but reduces drastically (0% – 6%) upon introduction of cholesterol in the lipid bilayer. The error bars in (g) and (h) are from the global fit.

Median distances between the membrane and C-terminal end of the protein from smFRET experiments.

The distance values were extracted from the distributions shown in Fig. 3b in the main text. The numbers in parenthesis indicates the 95 % confidence interval for experiments. Asterisk (*) indicates distance was beyond the FRET range for the snap surface 594 and cy5 dye pair (Ro = 8.4 nm).61

Median distances between the membrane and C-terminal end of the protein from smFRET experiments.

The distance values were extracted from the distributions shown in Fig. 3d and 3f in the main text. The numbers in parenthesis indicates the 95 % confidence interval for experiments. Asterisk (*) indicates distance was beyond the FRET range for the snap surface 594 and cy5 dye pair (Ro = 8.4 nm).61

Membrane composition influences EGFR conformation and function.

(Top) In healthy cells, EGF binding promotes transmembrane conformational coupling between the extracellular and intracellular domains of EGFR, enabling kinase activation through established signaling mechanisms. (Middle) Our results demonstrate that in membranes with high anionic lipid content, electrostatic repulsion between the negatively charged lipids and the kinase domain overrides EGF-induced transmembrane conformational coupling. (Bottom) Our results demonstrate that in cholesterol-rich membranes, increased membrane rigidity overrides EGF-induced transmem-brane conformational coupling. These findings establish membrane composition as a dominant regulator of EGFR signaling, independent of EGF binding.

Domains of EGFR.

EGFR consists of a 621-amino acid extracellular region (ED), a 24-amino acid transmembrane-spanning domain (TM), and an intracellular region, which is a 37-amino acid juxtamembrane domain (JM), a 273-amino acid kinase domain (KD) and a 231-amino acid C-terminal tail (CTT). The JM is further divided into juxtamembrane-A (JM-A) and juxtamembrane-B (JM-B) domains. Residues 978–990 are defined as N-terminal portion of the CTT (N-CTT) and residues 1070–1186 are defined as the C-terminal portion of the CTT (C-CTT). Residue numbering corresponds to EGFR excluding the signal sequence.

Production and characterization of full-length EGFR in nanodiscs.

(a) Cell-free reaction for the production of EGFR and nanodisc belt protein. Codon optimized DNA of the receptor protein (EGFR-SNAP) and the belt protein (ApoA1,6.49) are incubated together with lipid vesicles with or without cholesterol and E. Coli lysate at 25o C. (b) Stain-free (left) and fluorescence (right) gel images of the His-tag purified sample show the presence of ApoA1,6.49 at 25 kDa and full-length EGFR at 160 kDa, which implies successful EGFR production and insertion into nanodiscs. The presence of the EGFR band alone in the fluorescence gel image indicates successful and specific labeling. (c) SDS-PAGE analysis of His-tag purified ApoA1,6.49 (1 µg) produced in cell-free reaction. The two lanes correspond to replicate samples under identical conditions. (d) Western blots were performed on labelled EGFR in nanodiscs. Anti-EGFR Western blots (left) and anti-phosphotyrosine Western blots (right) tested the presence of EGFR and its ability to undergo tyrosine phosphorylation, respectively, consis-tent with previous experiments on similar preparations.18,54,55 The two lanes in each blot correspond to replicate samples under identical conditions.

Optimization of ApoA1 and EGFR co-expression in cell-free reactions.

Cell-free reactions were performed with different template rations of ApoA1,6.49:EGFR (1:2 to 1:200) and sampled at various time points (2 - 19 hours). Labeled lysines were incorporated during synthesis, ensuring that only proteins expressed in the cell-free reaction were fluorescently labeled. EGFR (160 kDa) and ApoA1,6.49 (25 kDa) expressions were monitored by SDS-PAGE.

Characterization of EGFR-containing nanodiscs in different anionic membrane environments.

(a) Dynamic light scattering (DLS) of EGFR in 100% POPC nanodiscs in PBS buffer indicates ∼38 nm average size. (b) Jitter plot showing the size distribution of EGFR in 100% POPC nanodiscs from negative-stain transmission electron microscopy (TEM). The horizontal line represents the mean size (33.4 ± 8.5 nm, N = 134). A representative TEM image is shown below. (c) DLS of EGFR in 85% POPC, 15% POPS nanodiscs in PBS buffer indicates ∼40.7 nm average size. (d) Jitter plot showing the size distribution of EGFR in 85% POPC, 15% POPS nanodiscs from negative-stain TEM. The horizontal line represents the mean size (36.8 ± 6.0 nm, N = 116). A representative TEM image is shown below. (e) DLS of EGFR in 70% POPC, 30% POPS nanodiscs in PBS buffer indicates ∼54.8 nm average size. (f) Jitter plot showing the size distribution of EGFR in 70 % POPC, 30 % POPS nanodiscs from negative-stain TEM. The horizontal line represents the mean size (37.8 ± 9.7 nm, N = 223). A representative TEM image is shown below. (g) DLS of EGFR in 40 % POPC, 60 % POPS nanodiscs in PBS buffer indicates ∼46.2 nm average size. (h) Jitter plot showing the size distribution of EGFR in 40 % POPC, 60 % POPS nanodiscs from negative-stain TEM. The horizontal line represents the mean size (41.2 ± 9.7 nm, N = 133). A representative TEM image is shown below.

Characterization of EGFR-containing nanodiscs in membrane environments containing cholesterol.

(a) Dynamic light scattering (DLS) of EGFR in 92.5% POPC, 7.5% cholesterol containing nanodiscs in PBS buffer indicates indicates 32.8 nm average size. (b) Jitter plot showing the size distribution of EGFR in 92.5% POPC, 7.5% cholesterol nanodiscs from negative-stain transmission electron microscopy (TEM). The horizontal line represents the mean size (33.6 ± 7.7 nm, N = 154). A representative TEM image is shown below. (c) DLS of EGFR in 80% POPC, 20% cholesterol containing nanodiscs in PBS buffer indicates 39.6 nm average size. (d) Jitter plot showing the size distribution of EGFR in 80% POPC, 20% cholesterol nanodiscs from TEM. The horizontal line represents the mean size (39.8 ± 5.9 nm, N = 188). A representative TEM image is shown below. (e) DLS of EGFR in EGFR in 62.5% POPC, 30% POPS, 7.5% cholesterol nanodiscs in PBS buffer indicates 33 nm average size. (f) Jitter plot showing the size distribution of EGFR in 62.5% POPC, 30% POPS, 7.5% cholesterol nanodiscs from TEM. The horizontal line represents the mean size (36.5± 7.8 nm, N = 159). A representative TEM image is shown below. (g) DLS of EGFR in 50% POPC, 30% POPC, 20% cholesterol nan-odiscs in PBS buffer indicates 39.2 nm average size. (h) Jitter plot showing the size distribution of EGFR in 50% POPC, 30% POPC, 20% cholesterol nanodiscs from TEM. The horizontal line represents the mean size (39.2± 6.3 nm, N = 281). A representative TEM image is shown below.

Characterization of anionic content in EGFR-embedded nanodiscs with zeta potential.56

Zeta potential distributions for EGFR in nanodiscs containing increasing amounts of anionic lipids (0%, 15%, 30% and 60% POPS). Maximum values from the distributions are shown in Fig. 1b in the main text. Error bars are from three technical replicates.

Characterization of cholesterol (Ch) content in EGFR-embedded nanodiscs with Laurdan.

Ensemble fluorescence excitation spectra (λem = 440 nm) of EGFR embedded Laurdan containing nanodiscs with 0% cholesterol, 7.5% cholesterol and 20% cholesterol. In the Laurdan excitation spectra, increase in the excitation band centered around 390 nm is observed with the addition of increasing amounts of cholesterol to the EGFR embedded nanodisc.57

Characterization of ss594 labeled EGFR nanodiscs in different anionic lipid environments.

(a) Ensemble fluorescence excitation spectra (λem = 660 nm) of ss594 labeled EGFR nanodiscs with 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. (b) Ensemble fluorescence emission spectra (λexc = 565 nm) of ss594 labeled EGFR nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. (c) Ensemble time-correlated single photon counting measurements for ss594 labeled EGFR nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. The instrument response function (IRF) is shown in gray.

Characterization of cy5 labeled EGFR nanodiscs in different anionic lipid environments.

(a) Ensemble fluorescence excitation spectra (λem = 700 nm) of cy5 labeled nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. (b) Ensemble fluorescence emission spectra (λex = 630 nm) of cy5 labeled nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. (c) Ensemble time-correlated single photon counting measurements were performed for cy5 labeled nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS. The instrument response function (IRF) is shown in gray.

Single ss594 labeled EGFR embedded nanodiscs.

(a) Confocal fluorescence image of immobilized constructs of EGFR in nanodiscs labeled with ss594 (λexc = 550 nm). (b) Representative intensity time trace from a single construct. The number of detected photons for each 100 ms interval was calculated and used to generate a fluorescence intensity trace (green) with the average intensity for the emissive period overlaid (black). (c) Histogram of the arrival times of detected photons generates the donor lifetime decay profile. Representative decay profiles of EGFR (green) with fit curve (black). The instrument response function (IRF) is shown in gray.

Jitter plots of the donor lifetime distributions from smFRET experiments to measure the distance between the ATP binding site and the C-terminus of EGFR.

Distributions of the donor lifetime from the histograms in Fig. 2j, k of main text are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the P-values (Supplementary Table 1). The median value of the donor only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.

Single Cy5 labeled EGFR embedded nanodiscs.

(a) Confocal fluorescence image of immobilized constructs of EGFR in nanodiscs containing a labeled Cy5 lipid (λexc = 640 nm). (b) Representative intensity time trace from a single construct. The number of detected photons for each 100 ms interval was calculated and used to generate a fluorescence intensity trace (red) with the average intensity for the emissive period overlaid (black).

Jitter plots of the donor lifetime distributions from smFRET experiments to measure the distance between the membrane and the C-terminus of EGFR.

Distributions of the donor lifetime from the histograms in Fig. 3b of main text are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the P-values (Supplementary Table 3). NS, not significant. The median value of the donor only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.

EGFR intracellular domain conformation is correlated with ATP binding.

(a) Correlation analysis between distance of EGFR C-terminus from the bilayer and extent of ATP binding in the (top) absence of EGF (Pearson correlation R = 0.75) and (bottom) presence of EGF (Pearson correlation R = 0.88).

Jitter plots of the donor lifetime distributions from smFRET experiments to measure the distance between the membrane and the C-terminus of EGFR.

Distributions of the donor lifetime from the histograms in Fig. 3d of main text are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the P-values (Supplementary Table 7). NS, not significant. The median value of the donor only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.

Jitter plots of the donor lifetime distributions from smFRET experiments to measure the distance between the membrane and the C-terminus of EGFR.

Distributions of the donor lifetime from the histograms in Fig. 3f of main text are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the P-values (Supplementary Table 10). NS, not significant. The median value of the donor only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.

smFRET donor lifetime distributions in DMPC and POPC nanodiscs.

smFRET donor fluorescence lifetime distributions in (a) 100% DMPC and (b) 100% POPC without EGF (top); with 1 µM EGF (bottom). Distributions of the donor lifetime from the histograms on the left in 100% DMPC (c) and 100% POPC (d) are represented as jitter plots along with the donor-only samples. The horizontal line indicates the median lifetime. One-way ANOVA was performed to obtain the P-values (Supplementary Table 3). The median value of the donor only distribution in each lipid environment was used as the reference value for calculations of the donor-acceptor distances for all smFRET measurements in that environment.

Statistical analysis of smFRET lifetime distributions.

Results from one-way analysis of variance (ANOVA) with P-value, F-statistic and degrees of freedom for all experimental pairs (experiment 1 and experiment 2 in the above table) from the distributions reported in Fig. 2j, k in the main text.

Sample sizes for smFRET measurements.

The number of molecules and number of photon bunches used to construct the lifetime distributions are reported for all smFRET histograms from Fig. 2j, k in the main text.

Statistical analysis of smFRET lifetime distributions.

Results from one-way analysis of variance (ANOVA) with P-value, F-statistic and degrees of freedom for all experimental pairs (experiment 1 and experiment 2 in the above table) from the distributions reported in Fig. 3b in the main text and Supplementary Fig. 17.

Sample sizes for smFRET measurements.

The number of molecules and number of photon bunches used to construct the lifetime distributions are reported for all smFRET histograms from Fig. 3b in the main text and Supplementary Fig. 17.

Model selection and statistical analysis of global fits.

Comparison of two- and three-Gaussian models used to globally fit the lifetime distributions across all experimental conditions. Parameters (µ, o) correspond to the mean and standard deviation of each Gaussian component, respectively. Likelihood, Bayesian Information Criterion (BIC), and Ashman’s D values are reported for assessing model quality and separation of components. The number of free parameters is 5 for the two-Gaussian model and 8 for the three-Gaussian model. The number of single-molecule photon bunches = 9,483 across all 18 conditions (lipid composition and ±EGF).

Amplitude of compact and open state of the EGFR intracellular domain from smFRET lifetime distributions in main text Fig. 3b.

The numbers in parenthesis indicates the error bar indicated in Fig. 3g.

Statistical analysis of smFRET lifetime distributions.

Results from one-way analysis of variance (ANOVA) with P-value, F-statistic and degrees of freedom for all experimental pairs (experiment 1 and experiment 2 in the above table) from the distributions reported in Fig. 3d in the main text.

Sample sizes for smFRET measurements.

The number of molecules and number of photon bunches used to construct the lifetime distributions are reported for all smFRET histograms from Fig. 3d in the main text.

Amplitude of compact and open state of the EGFR intracellular domain from smFRET lifetime distributions in main text Fig. 3d.

The numbers in parenthesis indicates the error bar indicated in Fig. 3g.

Statistical analysis of smFRET lifetime distributions.

Results from one-way analysis of variance (ANOVA) with P-value, F-statistic and degrees of freedom for all experimental pairs (experiment 1 and experiment 2 in the above table) from the distributions reported in Fig. 3f in the main text.

Sample sizes for smFRET measurements.

The number of molecules and number of photon bunches used to construct the lifetime distributions are reported for all smFRET histograms from Fig. 3f in the main text.

Amplitude of compact and open state of the EGFR intracellular domain from smFRET lifetime distributions in main text Fig. 3f.

The numbers in parenthesis indicates the error bar indicated in Fig. 3g.

List of antibodies used to show phosphorylation of EGFR nanodiscs.