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Membrane interaction of bound ligands contributes to the negative binding cooperativity of the EGF receptor.

Arkhipov A, Shan Y, Kim ET, Shaw DE - PLoS Comput. Biol. (2014)

Bottom Line: This cooperativity is widely believed to be central to the effects of ligand concentration on EGFR-mediated intracellular signaling.Although the extracellular portion of the human EGFR dimer has been resolved crystallographically, the crystal structures do not reveal the structural origin of this negative cooperativity, which has remained unclear.Here we report the results of molecular dynamics simulations suggesting that asymmetrical interactions of the two binding sites with the membrane may be responsible (perhaps along with other factors) for this negative cooperativity.

View Article: PubMed Central - PubMed

Affiliation: D. E. Shaw Research, New York, New York, United States of America.

ABSTRACT
The epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, migration, and differentiation, and aberrant EGFR signaling is implicated in a variety of cancers. EGFR signaling is triggered by extracellular ligand binding, which promotes EGFR dimerization and activation. Ligand-binding measurements are consistent with a negatively cooperative model in which the ligand-binding affinity at either binding site in an EGFR dimer is weaker when the other site is occupied by a ligand. This cooperativity is widely believed to be central to the effects of ligand concentration on EGFR-mediated intracellular signaling. Although the extracellular portion of the human EGFR dimer has been resolved crystallographically, the crystal structures do not reveal the structural origin of this negative cooperativity, which has remained unclear. Here we report the results of molecular dynamics simulations suggesting that asymmetrical interactions of the two binding sites with the membrane may be responsible (perhaps along with other factors) for this negative cooperativity. In particular, in our simulations the extracellular domains of an EGFR dimer spontaneously lay down on the membrane in an orientation in which favorable membrane contacts were made with one of the bound ligands, but could not be made with the other. Similar interactions were observed when EGFR was glycosylated, as it is in vivo.

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The two-ligand ectodomain dimer remaining symmetric in the simulations.(A) The two subunits of the ectodomain dimer, as observed at the end of one of the simulations without glycosylation (left) and at the end of the simulation with full glycosylation (right), superimposed using the Cα atoms of domains I–III for reference. The EGFR ectodomains I–III are shown in blue and red, and the EGF molecules bound to them are shown in yellow and orange, respectively. (B) Domain II maintaining the same conformation in both subunits of the two-ligand EGFR dimer. Angle Φ (the angle formed by Cα atoms of residues 194, 239, and 296 in EGFR, or 189, 235, and 289 in dEGFR) characterizes the bending of domain II. This angle is different in each of the two subunits of the asymmetric two-ligand dEGFR dimer (the solid and dashed black lines; PDB entry 3LTF). The average angles in the simulations of two- and one-ligand EGFR dimers (Figs. 2, 3, and 5; labels refer to nonglycosylated EGFR simulations 1, 2, and 3, and glycosylated EGFR simulation G) are shown for each of the two subunits in blue and red (error bars correspond to the standard deviation). The angle Φ is illustrated in the schematic of domains I, II, and III of EGFR on the right.
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pcbi-1003742-g004: The two-ligand ectodomain dimer remaining symmetric in the simulations.(A) The two subunits of the ectodomain dimer, as observed at the end of one of the simulations without glycosylation (left) and at the end of the simulation with full glycosylation (right), superimposed using the Cα atoms of domains I–III for reference. The EGFR ectodomains I–III are shown in blue and red, and the EGF molecules bound to them are shown in yellow and orange, respectively. (B) Domain II maintaining the same conformation in both subunits of the two-ligand EGFR dimer. Angle Φ (the angle formed by Cα atoms of residues 194, 239, and 296 in EGFR, or 189, 235, and 289 in dEGFR) characterizes the bending of domain II. This angle is different in each of the two subunits of the asymmetric two-ligand dEGFR dimer (the solid and dashed black lines; PDB entry 3LTF). The average angles in the simulations of two- and one-ligand EGFR dimers (Figs. 2, 3, and 5; labels refer to nonglycosylated EGFR simulations 1, 2, and 3, and glycosylated EGFR simulation G) are shown for each of the two subunits in blue and red (error bars correspond to the standard deviation). The angle Φ is illustrated in the schematic of domains I, II, and III of EGFR on the right.

Mentions: A simulation study of EGFR [22] has previously suggested that ectodomain interactions with the membrane may be at the root of the observed negative cooperativity of ligand binding. It was further suggested that the negative cooperativity may arise from the ectodomain's transition to a dEGFR-like asymmetric conformation, induced by interactions with the membrane. Although our simulations also suggest the important role of EGFR ectodomain interactions with the membrane, our simulations did not show a robust transition from a symmetric to an asymmetric conformation in the ectodomain dimer. Fig. 4A shows that, other than minor deviations due to the inherent flexibility of the loop regions, the dimer's two ectodomain subunits were nearly conformationally identical in our simulations. In particular, the conformations of domain II in the two subunits are highly similar, whereas in the dEGFR dimer the domain II is straight in one subunit and bent in the other, which ultimately leads to the different conformations of the two binding sites. This is illustrated in Fig. 4B, where the angle characterizing the bending of domain II is plotted. The angles of the two EGFR subunits were approximately the same in our simulations of the two-ligand dimer, much as they are in crystal structures [18], [19]. Our MM/GBVI calculation supports the notion that the two receptors of the two-ligand EGFR dimer maintain similar binding-site conformations while resting on the membrane: The two ligands have comparable MM/GBVI interaction energies with the receptors (Fig. 5B), including cases in which the receptors are glycosylated (Fig. 2C).


Membrane interaction of bound ligands contributes to the negative binding cooperativity of the EGF receptor.

Arkhipov A, Shan Y, Kim ET, Shaw DE - PLoS Comput. Biol. (2014)

The two-ligand ectodomain dimer remaining symmetric in the simulations.(A) The two subunits of the ectodomain dimer, as observed at the end of one of the simulations without glycosylation (left) and at the end of the simulation with full glycosylation (right), superimposed using the Cα atoms of domains I–III for reference. The EGFR ectodomains I–III are shown in blue and red, and the EGF molecules bound to them are shown in yellow and orange, respectively. (B) Domain II maintaining the same conformation in both subunits of the two-ligand EGFR dimer. Angle Φ (the angle formed by Cα atoms of residues 194, 239, and 296 in EGFR, or 189, 235, and 289 in dEGFR) characterizes the bending of domain II. This angle is different in each of the two subunits of the asymmetric two-ligand dEGFR dimer (the solid and dashed black lines; PDB entry 3LTF). The average angles in the simulations of two- and one-ligand EGFR dimers (Figs. 2, 3, and 5; labels refer to nonglycosylated EGFR simulations 1, 2, and 3, and glycosylated EGFR simulation G) are shown for each of the two subunits in blue and red (error bars correspond to the standard deviation). The angle Φ is illustrated in the schematic of domains I, II, and III of EGFR on the right.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109842&req=5

pcbi-1003742-g004: The two-ligand ectodomain dimer remaining symmetric in the simulations.(A) The two subunits of the ectodomain dimer, as observed at the end of one of the simulations without glycosylation (left) and at the end of the simulation with full glycosylation (right), superimposed using the Cα atoms of domains I–III for reference. The EGFR ectodomains I–III are shown in blue and red, and the EGF molecules bound to them are shown in yellow and orange, respectively. (B) Domain II maintaining the same conformation in both subunits of the two-ligand EGFR dimer. Angle Φ (the angle formed by Cα atoms of residues 194, 239, and 296 in EGFR, or 189, 235, and 289 in dEGFR) characterizes the bending of domain II. This angle is different in each of the two subunits of the asymmetric two-ligand dEGFR dimer (the solid and dashed black lines; PDB entry 3LTF). The average angles in the simulations of two- and one-ligand EGFR dimers (Figs. 2, 3, and 5; labels refer to nonglycosylated EGFR simulations 1, 2, and 3, and glycosylated EGFR simulation G) are shown for each of the two subunits in blue and red (error bars correspond to the standard deviation). The angle Φ is illustrated in the schematic of domains I, II, and III of EGFR on the right.
Mentions: A simulation study of EGFR [22] has previously suggested that ectodomain interactions with the membrane may be at the root of the observed negative cooperativity of ligand binding. It was further suggested that the negative cooperativity may arise from the ectodomain's transition to a dEGFR-like asymmetric conformation, induced by interactions with the membrane. Although our simulations also suggest the important role of EGFR ectodomain interactions with the membrane, our simulations did not show a robust transition from a symmetric to an asymmetric conformation in the ectodomain dimer. Fig. 4A shows that, other than minor deviations due to the inherent flexibility of the loop regions, the dimer's two ectodomain subunits were nearly conformationally identical in our simulations. In particular, the conformations of domain II in the two subunits are highly similar, whereas in the dEGFR dimer the domain II is straight in one subunit and bent in the other, which ultimately leads to the different conformations of the two binding sites. This is illustrated in Fig. 4B, where the angle characterizing the bending of domain II is plotted. The angles of the two EGFR subunits were approximately the same in our simulations of the two-ligand dimer, much as they are in crystal structures [18], [19]. Our MM/GBVI calculation supports the notion that the two receptors of the two-ligand EGFR dimer maintain similar binding-site conformations while resting on the membrane: The two ligands have comparable MM/GBVI interaction energies with the receptors (Fig. 5B), including cases in which the receptors are glycosylated (Fig. 2C).

Bottom Line: This cooperativity is widely believed to be central to the effects of ligand concentration on EGFR-mediated intracellular signaling.Although the extracellular portion of the human EGFR dimer has been resolved crystallographically, the crystal structures do not reveal the structural origin of this negative cooperativity, which has remained unclear.Here we report the results of molecular dynamics simulations suggesting that asymmetrical interactions of the two binding sites with the membrane may be responsible (perhaps along with other factors) for this negative cooperativity.

View Article: PubMed Central - PubMed

Affiliation: D. E. Shaw Research, New York, New York, United States of America.

ABSTRACT
The epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, migration, and differentiation, and aberrant EGFR signaling is implicated in a variety of cancers. EGFR signaling is triggered by extracellular ligand binding, which promotes EGFR dimerization and activation. Ligand-binding measurements are consistent with a negatively cooperative model in which the ligand-binding affinity at either binding site in an EGFR dimer is weaker when the other site is occupied by a ligand. This cooperativity is widely believed to be central to the effects of ligand concentration on EGFR-mediated intracellular signaling. Although the extracellular portion of the human EGFR dimer has been resolved crystallographically, the crystal structures do not reveal the structural origin of this negative cooperativity, which has remained unclear. Here we report the results of molecular dynamics simulations suggesting that asymmetrical interactions of the two binding sites with the membrane may be responsible (perhaps along with other factors) for this negative cooperativity. In particular, in our simulations the extracellular domains of an EGFR dimer spontaneously lay down on the membrane in an orientation in which favorable membrane contacts were made with one of the bound ligands, but could not be made with the other. Similar interactions were observed when EGFR was glycosylated, as it is in vivo.

Show MeSH
Related in: MedlinePlus