A molecular mechanism for the generation of ligand-dependent differential outputs by the epidermal growth factor receptor
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Evaluation Summary:
By revealing different conformational landscapes of EGFR when responding to different types of ligands, this study significantly advances our understanding the structural basis for how EGFR generates distinct downstream signaling in response to different types of ligands. This study represents an important advance in the field and paves the way for the comprehensive understanding of structural mechanisms underlying biased agonism in EGFR and other RTKs.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor-α (TGF-α), to the initiation of intracellular signaling pathways. EGFR binds to EGF and TGF-α with similar affinity, but generates different signals from these ligands. To address the mechanistic basis of this phenomenon, we have carried out cryo-EM analyses of human EGFR bound to EGF and TGF-α. We show that the extracellular module adopts an ensemble of dimeric conformations when bound to either EGF or TGF-α. The two extreme states of this ensemble represent distinct ligand-bound quaternary structures in which the membrane-proximal tips of the extracellular module are either juxtaposed or separated. EGF and TGF-α differ in their ability to maintain the conformation with the membrane-proximal tips of the extracellular module separated, and this conformation is stabilized preferentially by an oncogenic EGFR mutation. Close proximity of the transmembrane helices at the junction with the extracellular module has been associated previously with increased EGFR activity. Our results show how EGFR can couple the binding of different ligands to differential modulation of this proximity, thereby suggesting a molecular mechanism for the generation of ligand-sensitive differential outputs in this receptor family.
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Evaluation Summary:
By revealing different conformational landscapes of EGFR when responding to different types of ligands, this study significantly advances our understanding the structural basis for how EGFR generates distinct downstream signaling in response to different types of ligands. This study represents an important advance in the field and paves the way for the comprehensive understanding of structural mechanisms underlying biased agonism in EGFR and other RTKs.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Huang et al. reports the cryo-EM structures of EGF and TGFalpha bound to full-length EGFR. As for other receptor tyrosine kinases like the insulin and IGF1 receptors, the transmembrane helices and cytoplasmic kinase domains are not resolved in the cryo-EM maps.
3D classification of EGF-EGFR revealed multiple, closely-related conformational states of the ligand-bound ectodomain, in which a "scissor-like" rotation of the EGF binding portion of the ectodomain (D1-3) was correlated with a separation of the ends of the membrane-proximal domain (D4); the larger the scissor angle (~25{degree sign}), the closer the ends of D4 (~5 Å), and vice versa. For the smaller scissor angle of ~10{degree sign}, the two-fold symmetry of the EGF+EGFR complex breaks down, such that one of the D4 domains pivots …
Reviewer #1 (Public Review):
The manuscript by Huang et al. reports the cryo-EM structures of EGF and TGFalpha bound to full-length EGFR. As for other receptor tyrosine kinases like the insulin and IGF1 receptors, the transmembrane helices and cytoplasmic kinase domains are not resolved in the cryo-EM maps.
3D classification of EGF-EGFR revealed multiple, closely-related conformational states of the ligand-bound ectodomain, in which a "scissor-like" rotation of the EGF binding portion of the ectodomain (D1-3) was correlated with a separation of the ends of the membrane-proximal domain (D4); the larger the scissor angle (~25{degree sign}), the closer the ends of D4 (~5 Å), and vice versa. For the smaller scissor angle of ~10{degree sign}, the two-fold symmetry of the EGF+EGFR complex breaks down, such that one of the D4 domains pivots from D1-3 further than the other one, resulting in a D4 separation of ~20 Å.
The authors utilized previous NMR data on the isolated TM helices of EGFR, which indicated that there are two mutually exclusive crossovers points between the TM helices, one closer to the N-termini of the helices and one closer to the C-termini. Molecular dynamics simulations performed by the authors showed that, in general, the "tips-separated" configuration of the D4 domains was correlated with the N-terminal apposition of the TM helices, and the "tips-juxtaposed" configuration was correlated with the C-terminal apposition.
Previous biochemical data had indicated/suggested that the N-terminal dimerization of the TM helices results in higher kinase activity (through formation of the asymmetric kinase dimer) than the C-terminal dimerization, even though the C-terminal dimerization places the cytoplasmic juxtamembrane (JM) regions (leading into the kinase domains) closer together.
The authors determined cryo-EM structures of EGF bound to an EGFR mutant, L834R, which is a gain-of-function substitution in the activation loop of the kinase domain, and found that D4 in the tips-separated conformation was stabilized vs. in wild-type EGFR, indicating that a stabilized asymmetric kinase dimer is conformationally coupled to the tips-separated ectodomain conformation.
The authors determined cryo-EM structures with TGFalpha bound to EGFR and found that, in this ensemble of structures, D4 in the tips-separated conformation was destabilized (vs. in the EGF-EGFR structures) because of slight differences in the ligand-binding head of EGFR induced by TGFalpha vs. EGF binding.
All of these data - theirs and others - led to the hypothesis that EGF is a higher activity ligand than TGFalpha (despite both being high-affinity binders to EGFR) because of the conformational coupling between the ligand-binding head of EGFR, the distal tips of D4, the TM helices, the cytoplasmic JM region, and the asymmetric kinase dimer. To test this hypothesis, the authors performed in vitro and in-cell activity assays and, indeed, found that the level of EGFR phosphorylation was higher when stimulated with EGF vs. TGFalpha.
To provide evidence that the conformational coupling described above was responsible, the authors generated mutant EGFRs -a point mutation in D4 (W492G) and insertion in and replacement of the extracellular JM region - and measured phosphorylation levels upon stimulation with EGF or TGFalpha. These data showed that increasing the flexibility in these regions (through mutation) abrogated the phosphorylation difference in the two cases (EGF vs. TGFalpha), consistent with their hypothesis.
In summary, this is an impressive study providing solid evidence for a molecular mechanism by which two related, high-affinity growth factors, binding in exactly the same site, can achieve differential signaling outputs through a dimerized receptor tyrosine kinase, and represents an important advance in the field.
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Reviewer #2 (Public Review):
EGFR can be activated by several extracellular ligands. The molecular mechanisms of EGFR in differentiating extracellular signals from these ligands and transforming them into distinct intracellular signaling outputs are not fully understood. In this manuscript, Huang et al. carried out structural analysis of the full-length human EGFR (with ligand EGF or TGF-α) using cryo-EM and MD simulation. The authors reported that the dimeric structure of the two extracellular modules is not rigid at the dimeric interface, resulting in conformational fluctuations of individual domains. One interesting observation was the membrane-proximal tip of the extracellular module in representative two conformations, "separated" and "juxtaposed" states. The authors next tried to correlate the structural dynamics of EGFR to its …
Reviewer #2 (Public Review):
EGFR can be activated by several extracellular ligands. The molecular mechanisms of EGFR in differentiating extracellular signals from these ligands and transforming them into distinct intracellular signaling outputs are not fully understood. In this manuscript, Huang et al. carried out structural analysis of the full-length human EGFR (with ligand EGF or TGF-α) using cryo-EM and MD simulation. The authors reported that the dimeric structure of the two extracellular modules is not rigid at the dimeric interface, resulting in conformational fluctuations of individual domains. One interesting observation was the membrane-proximal tip of the extracellular module in representative two conformations, "separated" and "juxtaposed" states. The authors next tried to correlate the structural dynamics of EGFR to its signaling outputs.
Major concern:
Cryo-EM 3D classification was widely used to analyze the dynamics of protein complexes. It could be efficient in probing new intermediate states. But it is not strictly a quantitative method unless certain "unbiased" procedures are applied throughout the complete workflow of image processing. For example, the authors only showed classification results of one dataset (Figure 2). The particles appeared to be evenly distributed in the ten classes, which was a sign that the classification was not efficient and the resulting reconstructions are still mixtures of multiple states. To support the main conclusion of the manuscript, the authors should show the classification results of all datasets: (1) To our knowledge, cryoSparc is less efficient in separating particles into different conformations compared to Relion. The authors could try Relion and multiple rounds of 3D classification could be implemented. (2) Mask-based classification should be applied. (3) The authors could provide an analysis on the changes of particle distribution among different datasets.
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Reviewer #3 (Public Review):
This will be a landmark work in the RTK and EGFR fields. Huang et. al reported a series of cryo-EM structures of full-length EGFR/EGF complexes in different conformations. The major difference among these structures is the distance between the membrane proximal domains IV of EGFR. Although the TM and kinase domains of EGFR were not resolved in the cryo-EM maps, through comprehensive structural analysis and MD simulations, the authors proposed that, the EGFR/EGF complex with separated domains IV would induce N-terminal associated dimeric TM domain and high activity; whereas the EGFR/EGF complex with juxtaposed domains IV would promote C-terminal associated dimeric TM domain and low activity. Such claim is strongly supported by two structure evidences: (1) In the cryo-EM structure of EGFR L834R mutant/EGF …
Reviewer #3 (Public Review):
This will be a landmark work in the RTK and EGFR fields. Huang et. al reported a series of cryo-EM structures of full-length EGFR/EGF complexes in different conformations. The major difference among these structures is the distance between the membrane proximal domains IV of EGFR. Although the TM and kinase domains of EGFR were not resolved in the cryo-EM maps, through comprehensive structural analysis and MD simulations, the authors proposed that, the EGFR/EGF complex with separated domains IV would induce N-terminal associated dimeric TM domain and high activity; whereas the EGFR/EGF complex with juxtaposed domains IV would promote C-terminal associated dimeric TM domain and low activity. Such claim is strongly supported by two structure evidences: (1) In the cryo-EM structure of EGFR L834R mutant/EGF complex (a mutant that is supposed to have higher activity than EGFR WT), the separated domains IV is captured in a more stable state. (2) In the cryo-EM structure of EGFR with a weaker ligand TGF-a bound, the separated domains IV is in a more flexible conformation. In addition, the authors also introduced some mutations to EGFR, designed to break the structural coupling between domain IV and TM domain. These EGFR mutants can't response to EGF and TGF-a differently, which further supports the major conclusion of this work that the conformation of ECD determines the conformation of TM as well as the downstream signaling. Overall, the experiments were well designed, and the structural and functional works are of great quality.
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