SVEP1 enables efficient binding of Angiopoietin-2 to the TIE1 receptor, allowing receptor phosphorylation and downstream signaling
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eLife Assessment
This manuscript focuses on developing a structural model of how the multidomain ECM protein SVEP1 enables Angiopoietin (ANG) binding to the orphan receptor TIE1, resulting in downstream receptor phosphorylation and signaling. This is a potentially important study, however, it currently lacks key controls and is therefore incomplete. The data will be of interest to scientists working in vascular biology and RTK signaling.
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Abstract
Abstract
The molecular mechanisms that drive (lymph-)angiogenesis are crucial to understand diseases, such as lymphedema, that are caused due to malformations of the lymphatic vasculature. Recently an interaction between the secreted protein Svep1, a key regulator in lymphangiogenesis, and the transmembrane receptor Tie1 was shown in zebrafish, human and mice. Here, guided by in silico AlphaFold-multimer structure predictions of highly confident SVEP1 complexes, we assert with protein binding studies that the human CCP20 domain is the primary binding site for TIE1, and also show that in addition the CCP5-EGFL7 fragment of SVEP1 engages TIE1 as a secondary site. We further demonstrate that SVEP1 mediates strong binding of ANG2 and TIE1, and that combined stimulation of hdLECs with SVEP1 and ANG2 leads to phosphorylation of TIE1. TIE1 activation by SVEP1 and ANG2 enables downstream signaling and in turn potentiates nuclear exclusion of FOXO1 and phosphorylation of AKT compared to SVEP1 or ANG2 alone. We present a model where ANG1/2 dimers bind to both SVEP1 and TIE1, resulting in multiple TIE1 receptor molecules being recruited to a multimeric complex at the cell membrane, potentially amplifying its signaling capacity.
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eLife Assessment
This manuscript focuses on developing a structural model of how the multidomain ECM protein SVEP1 enables Angiopoietin (ANG) binding to the orphan receptor TIE1, resulting in downstream receptor phosphorylation and signaling. This is a potentially important study, however, it currently lacks key controls and is therefore incomplete. The data will be of interest to scientists working in vascular biology and RTK signaling.
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Reviewer #1 (Public review):
Summary:
In this manuscript, Uphoff et al. propose a structural and mechanistic model in which the multidomain ECM protein SVEP1 enables Angiopoietin (ANG) binding to the orphan receptor TIE1, thereby promoting downstream receptor phosphorylation and signaling. Using AlphaFold-based modeling, the authors predict that the CCP20 domain of SVEP1 binds to TIE1, creating a composite surface that facilitates Angiopoietin association and TIE1 activation. The resulting ternary model (SVEP1-TIE1-ANG) offers a structural rationale for how SVEP1 converts TIE1 into a functional, ligand-responsive receptor. Additional models and biological assays suggest roles for other domains of SVEP1, such as CCP5-EGF-L7, although these interactions are predicted with low confidence. The authors interpret these findings as the first …
Reviewer #1 (Public review):
Summary:
In this manuscript, Uphoff et al. propose a structural and mechanistic model in which the multidomain ECM protein SVEP1 enables Angiopoietin (ANG) binding to the orphan receptor TIE1, thereby promoting downstream receptor phosphorylation and signaling. Using AlphaFold-based modeling, the authors predict that the CCP20 domain of SVEP1 binds to TIE1, creating a composite surface that facilitates Angiopoietin association and TIE1 activation. The resulting ternary model (SVEP1-TIE1-ANG) offers a structural rationale for how SVEP1 converts TIE1 into a functional, ligand-responsive receptor. Additional models and biological assays suggest roles for other domains of SVEP1, such as CCP5-EGF-L7, although these interactions are predicted with low confidence. The authors interpret these findings as the first structural framework for how SVEP1 enables ANG-TIE1 signaling.
Strengths:
(1) The central hypothesis - that SVEP1 enables ANG binding to the orphan receptor TIE1 - is biologically compelling and addresses an important question in vascular biology.
(2) The AlphaFold-predicted ternary complex (SVEP1-TIE1-ANG) is plausible, high-confidence, and structurally consistent with prior functional data (e.g., poly-Ala scanning from Sato-Nishiuchi et al.).
(3) The authors' model offers a potential explanation for the previously observed role of SVEP1 in enhancing ANG signaling through TIE1, and may represent the first structural insight into TIE1's transition from orphan to ligand-activated receptor.
(4) The potential clinical implication - that a combinatorial ligand (ANG+SVEP1) can activate TIE1- could have translational relevance for vascular leak and inflammatory disease.
Weaknesses:
(1) Lack of structural validation and mechanistic follow-up: Despite the promising AlphaFold model, there are no figures of the predicted interface, no residue-level interactions shown, no ipTM values reported, and no experimental follow-up to test the interface. PAE plots are incorrectly used as confidence justifications, which is not appropriate for complex predictions.
(2) Biophysical validation is missing: No surface plasmon resonance (SPR), ITC, or biochemical assays are included to confirm ternary complex formation or quantify binding kinetics. Given the manuscript's structural focus, this is a major gap. For instance, an SPR experiment where ANG is immobilized, and TIE1 binding is measured {plus minus} SVEP1, would directly test the model. And allow direct comparison to ANG-TIE2.
(3) Missed opportunity for mutagenesis-driven validation: The manuscript does not include any interface-targeted mutations, despite clear opportunities. For example, mutating T2595 in SVEP1 (to R) or mutating the TIE1-specific residues (residues PL 202-203 to LF) could strongly test the model and potentially reveal dominant-negative behaviors. E.g. A T2595 mutant should block ANG binding but not TIE1 binding.
(4) Overinterpretation of weak models: The additional AlphaFold model involving the CCP5-EGFL7 domains binding TIE1 has extremely low confidence (ipTM < 0.15) when reexamined by this reader and should not be emphasized. There is no biophysical evidence or binding data (SPR) to support this interaction, and its inclusion detracts from the much stronger CCP20 model.
(5) Language around modeling is overstated and potentially misleading: Terms like "unequivocal," "high-affinity," or "affirms strong binding" in reference to AlphaFold predictions are inappropriate. These are hypotheses -not confirmations - and must be tested at the biochemical level. This should be clarified throughout the manuscript to ensure non-experts do not misinterpret modeling confidence as binding affinity.
(6) Negative stain EM data is not informative due to low resolution and lack of defined interfaces; unless replaced by higher-resolution Cryo-EM, this should be omitted. Better would be co-gel filtration, AUC, or SEC-MALLs with ANG-SVEP1-TIE1.
(7) Disjointed narrative: The manuscript presents a compelling mechanism involving CCP20-driven ANG binding to TIE1, but then becomes fragmented by introducing the low-confidence CCP5-EGFL7 model and speculative higher-order polymerization models that are not experimentally supported.
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Reviewer #2 (Public review):
Uphoff and colleagues present the results of a study focused on characterizing the binding of SVEP1 to TIE1 along with Angiopoietin-2. Starting with computational prediction of SVEP1 binding to TIE1, the authors identify the region of SVEP1 that serves as a high-affinity ligand for TIE1. Advanced studies identify a weak secondary binding site within SVEP1 that appears to be sufficient but not necessary for its interaction with TIE1 based on in vivo rescue experiments. The most novel contribution of the manuscript seems to be the identification of angiopoietin-1 and -2 as co-factors that seem to enhance the binding of SVEP1 with TIE1 and impact downstream AKT signaling. They propose a complex in which SVEP1 binds to TIE1 and ANG2.
Although the first set of results is essentially confirmatory, the …
Reviewer #2 (Public review):
Uphoff and colleagues present the results of a study focused on characterizing the binding of SVEP1 to TIE1 along with Angiopoietin-2. Starting with computational prediction of SVEP1 binding to TIE1, the authors identify the region of SVEP1 that serves as a high-affinity ligand for TIE1. Advanced studies identify a weak secondary binding site within SVEP1 that appears to be sufficient but not necessary for its interaction with TIE1 based on in vivo rescue experiments. The most novel contribution of the manuscript seems to be the identification of angiopoietin-1 and -2 as co-factors that seem to enhance the binding of SVEP1 with TIE1 and impact downstream AKT signaling. They propose a complex in which SVEP1 binds to TIE1 and ANG2.
Although the first set of results is essentially confirmatory, the identification of ANG-2 as a "co-factor" enhancing the binding of SVEP1 to TIE1 and associated downstream signaling (i.e., Figures 3 and 4) is novel and is of interest. However, the manuscript and its conclusions would greatly benefit from some clarifying details and additional experiments to ensure rigor and support specific claims.
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