Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

Abstract

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.

Figure 1

Solubility and monodispersity of recombinant EGFR in detergent-solubilized cell lysates. EGFR-GFP fusion protein was expressed by transient transfection in 293S GntI−/− cells and solubilized in buffer containing (solid line) 1% Triton X-100 or (dashed line) 1% octyl β-d-glucopyranoside. Lysates were subjected to gel filtration on Superose 6HR (24 mL bed volume). GFP fluorescence of eluate fractions was monitored at excitation and emission wavelengths of 488 and 512 nm, respectively.

Figure 2

EGF-dependent dimerization and kinase activation of purified, detergent-soluble EGFR. (a) Δ998-EGFR was expressed by transient transfection in 293S GntI−/− cells and purified from detergent-solubilized cell lysates by streptactin–Sepharose affinity chromatography in the presence of 0.1% Triton X-100. Lanes 1−5 show 5 μL of the corresponding 0.5 mL fractions eluted with 2.5 mM desthiobiotin and subjected to reducing SDS–PAGE and Coomassie blue staining. (b, c) Gel filtration chromatograms of EGFR samples from lane 3 in (a) treated (b) without or (c) with 20 μM EGF and subjected to gel filtration on Superose 6HR equilibrated in 0.4 mM dodecyl maltoside. Protein elution was monitored by absorbance at 280 nm (solid line), and kinase activity of eluate fractions was determined by monitoring the incorporation of ³²P into an exogenous peptide substrate (dashed line, circles).

Figure 3

Overall structure and domain organization of EGFR determined by negative stain electron microscopy. (a) Δ998-EGFR samples purified by streptactin–Sepharose affinity chromatography were treated with 4 μM EGF for 30 min on ice and subjected to gel filtration on Superose 6HR equilibrated in 0.4 mM dodecyl maltoside. Samples of the peak fraction corresponding to dimeric EGF-bound EGFR were imaged by negative stain EM and used to calculate class averages. The averages from the most populated classes are shown. (b) Masked region of density corresponding to the extracellular domain in (a) that was used for cross-correlation with two-dimensional projections calculated from crystal structures of the dimeric, liganded EGFR extracellular domain (c and d). Two-dimensional projections and cross-correlation coefficients from (c) the extracellular domain EGF-bound crystal structure that contains domains I–III and (d) the model of the entire extracellular domain including domain IV. (e) Ribbon diagram of the dimeric EGF-bound EGFR extracellular domain crystal structure containing domains I–III. (f) Model of the entire EGFR extracellular domain generated by aligning domain IV from the unliganded structure of the extracellular domain (14) with the first two Cys-rich modules of domain IV (residues 480−512) that were resolved in the liganded structure (17). The ribbon diagrams (e and f) are shown in approximately the same orientations as the two-dimensional projections shown in (c) and (d).

Figure 4

Stability of EGFR kinase activity in dodecyl maltoside. (a) Δ998-EGFR samples purified by streptactin–Sepharose affinity chromatography in the presence of 0.1% Triton X-100 were treated with EGF and subjected to gel filtration on Superose 6HR equilibrated in (filled circles, solid line) 0.4 mM dodecyl maltoside or (open circles, dashed line) 0.2 mM dodecyl maltoside. The peak fraction corresponding to dimeric EGF-bound EGFR was dispensed into aliquots and frozen at −80 °C. Samples were thawed and stored on ice for the indicated amount of time and assayed for kinase activity by monitoring the incorporation of ³²P into an exogenous peptide substrate. (b) Peak fractions corresponding to Δ998-EGFR dimer species eluted from gel filtration in 0.2 mM dodecyl maltoside were refractionated by gel filtration either (top) immediately or (bottom) following incubation on ice for 10 h. (c) Kinase activity of the purified EGFR kinase domain (residues 672−998) in the (open circles, dashed line) absence or (filled circles, solid line) presence of 0.2 mM dodecyl maltoside. (d) Kinase activity of Δ998-EGFR in the presence of 0.2 mM dodecyl maltoside and brain whole lipid extract at detergent:lipid weight ratios of (circles, solid line) 4:1, (squares, dashed line) 1:1, and (triangles, dashed-dotted line) 1:10.

Figure 5

Reconstitution of EGFR dimers in phospholipid nanodiscs. Phospholipid nanodiscs were assembled in the (a) absence or (b) presence of purified WT-EGFR pretreated with EGF. Assembly mixtures consisted of 10 μM membrane scaffold protein MSP1T2 and 750 μM egg phosphatidylcholine without or with 1 μM EGFR plus 20 μM EGF. Disc formation and receptor incorporation were analyzed by gel filtration on Superose 6HR equilibrated in 10 mM Tris, pH 7.4, and 100 mM NaCl. Protein elution was monitored by absorbance at 280 nm (solid line), and kinase activity of eluate fractions was determined by monitoring the incorporation of ³²P into an exogenous peptide substrate (dashed line, circles). (c) Ni-NTA purification of EGFR-incorporated nanodiscs monitored by SDS–PAGE and Coomassie blue staining of input, flow-through, wash, and eluate fractions. The molar ratio of EGFR:MSP1T2(−) in eluate fractions was estimated by comparing the staining intensities of EGFR and MSP1T2(−) bands relative to the staining intensities of the 150 and 20 kDa molecular mass standards, respectively.

Figure 6

Stability of EGFR reconstituted in phospholipid bilayer nanodiscs and vesicles. (a) Purified WT-EGFR reconstituted in phospholipid bilayer nanodiscs in the presence of EGF was assayed for structural stability by gel filtration (solid line) immediately following disc assembly or (dashed line) following a 48 h incubation at 4 °C. The absorbance at 280 nm for each trace is shown as a relative value with respect to the peak in absorbance that elutes at 15.51 mL to account for different sample volumes injected on the gel filtration column. (b, c) The functional stability of Δ998-EGFR reconstituted in (b) phosphatidylcholine nanodiscs or (c) vesicles was assayed by monitoring tyrosine kinase activity as a function of time incubated at 4 °C following disc or vesicle assembly. (d) Specific activity of Δ998-EGFR reconstituted in 0.1% Triton X-100 or phosphatidylcholine nanodiscs measured after (black bars) 0 h or (gray bars) 24 h incubation at 4 °C. Note that 0 h for the samples reconstituted in nanodiscs is relative to once disc assembly has been completed.