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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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Simulations of the one-ligand dimer.(A) The conformation of the one-ligand ectodomain dimer obtained from a simulation employing the crystal structure of the two-ligand dimer (PDB entry 3NJP; ref. [23]), with the ligand removed from the red subunit, as a starting state. Domains I–IV and the EGF molecule are marked. The one-ligand dimer differs from the two-ligand dimer in the conformation of the domain IV of the red subunit. (B) The one-ligand dimer lying down on the membrane. The ligand bound to this dimer faces the membrane. Snapshots from simulations of the nonglycosylated and glycosylated dimers are shown. (C) The free energy of a ligand's interaction with its host receptor in a one-ligand EGFR dimer (upper panels) estimated using MM/GBVI, the strength of its interaction with the membrane bilayer (middle panels) estimated in the same way, and the distance between the ligand's N-terminus and the membrane (lower panels) in three independent simulations in which the receptors were not glycosylated and in one additional simulation in which they were. Also shown (middle panels) is the total surface area of the ligand buried due to its interactions with the receptor and the membrane. As indicated by these data, the additional free energy conferred by the ligand's membrane interaction is a significant fraction of its interaction energy with the receptor.
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pcbi-1003742-g003: Simulations of the one-ligand dimer.(A) The conformation of the one-ligand ectodomain dimer obtained from a simulation employing the crystal structure of the two-ligand dimer (PDB entry 3NJP; ref. [23]), with the ligand removed from the red subunit, as a starting state. Domains I–IV and the EGF molecule are marked. The one-ligand dimer differs from the two-ligand dimer in the conformation of the domain IV of the red subunit. (B) The one-ligand dimer lying down on the membrane. The ligand bound to this dimer faces the membrane. Snapshots from simulations of the nonglycosylated and glycosylated dimers are shown. (C) The free energy of a ligand's interaction with its host receptor in a one-ligand EGFR dimer (upper panels) estimated using MM/GBVI, the strength of its interaction with the membrane bilayer (middle panels) estimated in the same way, and the distance between the ligand's N-terminus and the membrane (lower panels) in three independent simulations in which the receptors were not glycosylated and in one additional simulation in which they were. Also shown (middle panels) is the total surface area of the ligand buried due to its interactions with the receptor and the membrane. As indicated by these data, the additional free energy conferred by the ligand's membrane interaction is a significant fraction of its interaction energy with the receptor.

Mentions: Because a crystal structure of a singly liganded ectodomain of an EGFR dimer is not yet available, we made a model based on the crystal structure of the two-ligand ectodomain dimer by removing one bound ligand from the crystal structure [26]. We here simulated the one-ligand ectodomain dimer in this conformation, connected with the TM segments, three times. In all three simulations, the ectodomain dimer, which was initially in an upright orientation, spontaneously lay down on the membrane (Fig. 3B), allowing the ligand to come into contact and develop extensive interactions with the membrane. We again calculated the MM/GBVI energy of the ligand's interaction with the membrane (Fig. 3C). The results suggest that the membrane interaction is energetically favorable, and that the free energy increase associated with the ligand's membrane interaction is a significant fraction of the free energy arising from its interaction with EGFR.


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)

Simulations of the one-ligand dimer.(A) The conformation of the one-ligand ectodomain dimer obtained from a simulation employing the crystal structure of the two-ligand dimer (PDB entry 3NJP; ref. [23]), with the ligand removed from the red subunit, as a starting state. Domains I–IV and the EGF molecule are marked. The one-ligand dimer differs from the two-ligand dimer in the conformation of the domain IV of the red subunit. (B) The one-ligand dimer lying down on the membrane. The ligand bound to this dimer faces the membrane. Snapshots from simulations of the nonglycosylated and glycosylated dimers are shown. (C) The free energy of a ligand's interaction with its host receptor in a one-ligand EGFR dimer (upper panels) estimated using MM/GBVI, the strength of its interaction with the membrane bilayer (middle panels) estimated in the same way, and the distance between the ligand's N-terminus and the membrane (lower panels) in three independent simulations in which the receptors were not glycosylated and in one additional simulation in which they were. Also shown (middle panels) is the total surface area of the ligand buried due to its interactions with the receptor and the membrane. As indicated by these data, the additional free energy conferred by the ligand's membrane interaction is a significant fraction of its interaction energy with the receptor.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003742-g003: Simulations of the one-ligand dimer.(A) The conformation of the one-ligand ectodomain dimer obtained from a simulation employing the crystal structure of the two-ligand dimer (PDB entry 3NJP; ref. [23]), with the ligand removed from the red subunit, as a starting state. Domains I–IV and the EGF molecule are marked. The one-ligand dimer differs from the two-ligand dimer in the conformation of the domain IV of the red subunit. (B) The one-ligand dimer lying down on the membrane. The ligand bound to this dimer faces the membrane. Snapshots from simulations of the nonglycosylated and glycosylated dimers are shown. (C) The free energy of a ligand's interaction with its host receptor in a one-ligand EGFR dimer (upper panels) estimated using MM/GBVI, the strength of its interaction with the membrane bilayer (middle panels) estimated in the same way, and the distance between the ligand's N-terminus and the membrane (lower panels) in three independent simulations in which the receptors were not glycosylated and in one additional simulation in which they were. Also shown (middle panels) is the total surface area of the ligand buried due to its interactions with the receptor and the membrane. As indicated by these data, the additional free energy conferred by the ligand's membrane interaction is a significant fraction of its interaction energy with the receptor.
Mentions: Because a crystal structure of a singly liganded ectodomain of an EGFR dimer is not yet available, we made a model based on the crystal structure of the two-ligand ectodomain dimer by removing one bound ligand from the crystal structure [26]. We here simulated the one-ligand ectodomain dimer in this conformation, connected with the TM segments, three times. In all three simulations, the ectodomain dimer, which was initially in an upright orientation, spontaneously lay down on the membrane (Fig. 3B), allowing the ligand to come into contact and develop extensive interactions with the membrane. We again calculated the MM/GBVI energy of the ligand's interaction with the membrane (Fig. 3C). The results suggest that the membrane interaction is energetically favorable, and that the free energy increase associated with the ligand's membrane interaction is a significant fraction of the free energy arising from its interaction with EGFR.

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