Structural insights into the activation of the chicken ROS1 receptor by the NEL/NICOL ligand complex

Domain: chemistry
Published: 2026-02-24
Editorial outcome: major_revision

Abstract
The receptor tyrosine kinase ROS1 plays essential roles in cell growth and sperm maturation, yet its activation mechanism has remained poorly understood. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of chicken ROS1 in its ligand-free form, in complex with its ligand NEL, and with the ligand/co-ligand complex NEL/NICOL. Unliganded ROS1 adopts an arc-shaped conformation. The interaction between NEL and ROS1 is mediated by the VWC2 domain of NEL and the β1 domain of ROS1. Binding of NICOL to the coiled-coil domain of NEL stabilizes NEL into a batwing-shaped asymmetric dimer, which can recruit only one ROS1 molecule due to steric hindrance. Structural analyses and biochemical results suggest that the 2:1 NEL/NICOL complexes further oligomerize through LamG–VWC4 domain interactions, facilitating the clustering of multiple ROS1 for its activation. Functional assays confirm that both NICOL and the multimerization of NEL/NICOL complexes are required for robust ROS1 signaling. Our findings establish NICOL as a critical co-ligand for ROS1 and suggest a distinct ligand-driven oligomerization mechanism for ROS1 activation.

Introduction
The ROS1 gene is evolutionarily conserved across a broad range of animal species, from early-diverging metazoans to mammals1. It encodes a receptor tyrosine kinase (RTK)2,3, a type of transmembrane receptor that plays vital roles in regulating animal growth and development. In higher vertebrate species, ROS1 is expressed in various tissues, including testis, lung, heart, intestine, and kidney4–7, and has been implicated in various physiological processes, including regulating growth and development1. Recent research has indicated that ROS1 signaling is required for sperm maturation and male fertility8. Aberrant expression, mutations, and somatic chromosomal fusions of the ROS1 gene have been implicated in a variety of diseases, including both cancerous and non-cancerous conditions1,9. Dysregulated ROS1 expression—such as overexpression, splice variants, and gene amplification—can drive oncogenesis across multiple tissues and organs, including the lung, breast, stomach, liver, kidney, and colon1,9. Notably, ROS1 frequently fuses with other oncogenes to form ROS1 fusion proteins, which act as potent oncogenic drivers10–14. Over 50 different 5′ gene partners have been identified that fuse with the 3′ region of ROS19. These fusion partners typically harbor dimerization domains, leading to constitutive, ligand-independent activation of ROS1’s kinase activity1,9,14. In addition to its oncogenic roles, mutations in ROS1 have also been associated with cardiovascular disorders1. ROS1 is the largest RTK identified to date2. Structurally, like other RTKs, ROS1 consists of an extracellular domain (ECD), a transmembrane (TM) helix, and an intracellular kinase domain (KD). The ROS1 ECD is predicted to include a cysteine-rich ALKAL-type coupled helices domain (CATCH), nine fibronectin type III domains (FnIII-1–FnIII-9), and three β-propeller domains (β1–β3) (Fig. 1a). Recent cryo-electron microscopy (cryo-EM) studies have reported structures of Sevenless (Sev), the Drosophila ortholog of vertebrate ROS115,16. These studies determined the structures of the ligand-free-Sev, the ligand (BOSS)-bound Sev, and dimeric Sev at low pH. Although these structures shed light on the three-dimensional domain organization of the ROS1 ECD, they offer limited insight into ligand recognition and signal transduction mechanisms of ROS1 in higher vertebrates.

Vertebrate ROS1 had long been considered an orphan receptor, until a study in 2020 reported that neural epidermal growth factor–like 2 (NELL2) specifically binds to the extracellular domain of mouse ROS18. This interaction was shown to activate the ERK signaling pathway, which is essential for the differentiation of the initial segment of the caput epididymis and ultimately critical for sperm maturation. Follow-up work by the same group identified NICOL (NELL2-interacting cofactor for lumicrine signaling), a small secreted protein, is necessary for ROS1/NELL2–mediated ERK activation17,18. However, the precise mechanism by which NELL2 and NICOL together activate ROS1 remains unclear.

Here, we determine the cryo-EM structures of the ligand-free form of chicken ROS1 (cROS1), the cROS1–chicken NEL (cNEL, homologous to human NELL2) complex, and the cROS1/cNEL/hNICOL complex. In its ligand-free state, cROS1 adopts an arc-shaped conformation. The binding of cNEL is mediated by the VWC2 domain of cNEL and the β1 domain of cROS1. The cNEL/hNICOL complex forms an asymmetric, batwing-like structure, in which the coiled-coil (CC) domains of the two cNEL protomers and α-helical region of hNICOL together form a central three-helix bundle. Despite the presence of two cNEL protomers in this complex, it can only engage one cROS1 molecule at a time due to steric hindrance. Further structural and functional analyses suggest that ROS1 activation occurs through higher-order oligomerization of the ROS1/NEL/NICOL complex, bringing multiple ROS1 receptors into proximity and enabling trans-autophosphorylation.

Results
Structural determination of chicken ROS1 in the ligand-free-state
To investigate the structure of vertebrate ROS1 in its unliganded state, we purified the full ECD of chicken ROS1 (cROS1), which exhibits high expression levels and shares 54% sequence identity with human ROS1 (Supplementary Fig. 1). Initial cryo-EM analysis of the cROS1-ECD revealed an arc-shaped architecture comprising two rigid parts, referred to as the head and leg regions respectively (Supplementary Fig. 2). Subsequent 3D classification identified a subtle hinge-motion between these two regions, with the angle between them ranging from 120° to 150° (Fig. 1b). Focused refinement enabled us to resolve the head and leg regions at resolutions of 2.9Å and 3.6Å, respectively, allowing accurate modeling of nearly the entire cROS1 ECD, spanning residues 24 to 1865 (Supplementary Fig. 2).

Overall architecture of ligand-free cROS1
The head region of cROS1 consists of the CATCH, the first four FnIII domains (FnIII-1 to FnIII-4), and the first two β-propeller domains (β1 and β2) (Fig. 1b, c). The CATCH is a domain not typically found in other RTKs and is composed of four α-helices stabilized by multiple disulfide bonds. Within the head, the β1–FnIII-3–β2–FnIII-4 segment folds back and aligns anti-parallelly with the CATCH–FnIII-1–FnIII-2 segment, forming a compact, “D”-shaped architecture (Fig. 1b, c). This conformation is stabilized by extensive electrostatic and hydrophobic interactions—specifically between FnIII-2 and β1–FnIII-3, as well as between the CATCH–FnIII-1 and β2 (Fig. 1c). Additionally, a loop from the CATCH domain inserts into a cavity formed by β2 and FnIII-4, further reinforcing the rigidity of the head structure (Fig. 1c). A similar D-shaped fold has been observed in the structure of Sevenless (Sev), the Drosophila ortholog of ROS1, indicating the structural conservation of ROS1 across species15,16.

In contrast to the compact head region, the leg region of cROS1—comprising the FnIII-5 to FnIII-9 domains and the β3 domain—adopts an extended, linear conformation spanning approximately 200Å (Fig. 1b). Within this region, the FnIII-5 domain packs closely against β3 but forms only a weak association with the adjacent FnIII-4 domain of the “head,” accounting for the observed flexibility between the head and leg regions of cROS1. Notably, several N-glycans are positioned at the interfaces between consecutive domains from FnIII-6 to FnIII-9, enhancing interdomain interactions and contributing to the high rigidity of the “leg” (Fig. 1c).

Structure determination and overall architecture of cROS1 in the cNEL bound state
To investigate the activation mechanism of ROS1 by NEL, we purified the chicken NEL protein (cNEL), which shares 88% sequence identity with human NELL2, using HEK293F cells. Purified cNEL primarily exists as a disulfide-linked dimer, with a small fraction present as a trimer19,20 (Supplementary Fig. 1). Interestingly, a previous study indicates that recombinant human NELL1 and NELL2 purified from insect cells predominantly exist as trimers21. The observed differences in oligomeric state may have arisen from differences in the expression system or from species-specific variation. We first analyzed the binding affinity between cNEL and cROS using biolayer interferometry (BLI). The results showed that cNEL binds cROS1 strongly, with the K value in the single-digit nanomolar range (~2 nM) (Supplementary Fig. 3). Subsequently, we reconstituted the cROS1/cNEL complex for cryo-EM structural analysis (Fig. 2a and Supplementary Figs. 1, 4, 5a). Co-elution of cROS1 and cNEL during size-exclusion chromatography (SEC) indicated the formation of a stable complex (Supplementary Fig. 1). Initial 3D reconstruction of the cROS1/cNEL complex was resolved at 2.6Å resolution, revealing the complete head region of cROS1 and the VWC2 domain of cNEL. Subsequent 3D classification allowed the visualization of the VWC1 domain and the first two EGF domains (EGF1–2) of cNEL, though at relatively low resolution (Fig. 2b, c and Supplementary Fig. 4).

Our cryo-EM model reveals that the VWC1–VWC2–EGF1–EGF2 domains adopt a pin-like architecture (Fig. 2c). VWC1 interacts directly with EGF1, stabilizing the pin-like fold, while VWC2 is positioned at the tip of the pin, facilitating its interaction with cROS1. The remainder of the cNEL structure was unresolved, likely due to substantial conformational flexibility. Comparative structural analysis between the cROS1/cNEL complex and ligand-free-cROS1 indicates that cROS1 does not undergo significant conformational changes upon complex formation with cNEL. Given that cNEL predominantly forms a flexible disulfide-linked dimer, a single cNEL dimer may be capable of binding two cROS1 molecules simultaneously, although such dimeric complex is not resolved in our cryo-EM analysis.

The interactions between cROS1 and cNEL
In the cROS1/cNEL complex, cNEL is positioned atop the head region of cROS1, with the interaction interface formed between a lateral surface of the β1 domain of cROS1 and a major β-sheet within the VWC2 domain of cNEL (Fig. 2b–d). Specifically, hydrophobic interactions between Phe336, Val349, Val357, and Phe359 of cNEL and Phe318, Leu319, and Val543 of cROS1 are critical for mediating the cROS1/cNEL association (Fig. 2d). Additionally, a salt bridge forms between Arg368 of cNEL and Glu541 and Asp545 of cROS1. Several other residues—Ser351, Lys365, and Gln367 of cNEL, along with Arg524, Phe527, Glu530, Ser538, Asn540, Pro551, and Tyr553 of cROS1—also contribute to the stability of the interface (Fig. 2d).

To validate the functional importance of this interface, we introduced F318A, E541A, F527A, and Y553A point mutations into the β1 domain of cROS1. These cROS1 mutants exhibited reduced activity in response to cNEL stimulation (Fig. 2e). Together, these findings underscore the critical role of the VWC2–β1 interface in cROS1 activation.

The cryo-EM structure of cROS1/cNEL/hNICOL1 complex
NICOL has been shown to enhance NEL-dependent ROS1 activation by directly associating with NEL17. To investigate the underlying mechanism, we co-expressed cNEL with either chicken NICOL (cNICOL) or human NICOL (hNICOL), which shares 72% sequence identity (Fig. 3a and Supplementary Fig. 5b), in HEK293 cells. The cNEL/hNICOL complex exhibited higher expression levels and better biochemical properties compared to the cNEL/cNICOL complex (Supplementary Figs. 6 and 7a). We therefore chose the cNEL/hNICOL complex for further characterization. SDS-PAGE analysis revealed that cNEL and hNICOL form a highly stable complex, which could only be disrupted by reducing agents such as dithiothreitol (DTT), indicating that the complex is stabilized by disulfide bonds (Supplementary Fig. 6). The cNEL/hNICOL complex potently activates cROS1, leading to enhanced ROS1 autophosphorylation and downstream ERK phosphorylation (Supplementary Figs. 7b and 8). In addition, our cell-based assay showed that the cNEL/hNICOL complex activates ROS1 to a level comparable to that of the cNEL/cNICOL complex (Supplementary Fig. 7c), suggesting that the species-mixed complex does not introduce unintended artifacts and is valid for the mechanistic analyses.

The BLI results showed that cROS1/cNEL and cROS1/cNEL/hNICOL have nearly identical binding affinities (Supplementary Fig. 3). Nevertheless, consistent with previous findings17, our cell-based assays demonstrated that the cNEL/hNICOL complex activates cROS1 much more robustly than cNEL alone (Fig. 2e). Moreover, dose-dependent ligand analyses showed that the cNEL/hNICOL complex induced robust cROS1 activation even at low concentrations, whereas cNEL alone failed to elicit strong activation, even at concentrations up to 2000 nM (Supplementary Fig. 9a). Time-course analysis further indicated that maximal cROS1 activation by either the cNEL/hNICOL complex or cNEL alone was reached at approximately 30 min, but the level of activation by cNEL alone was much lower (Supplementary Fig. 9b). These results suggest that cNEL alone is not optimal for cROS1 activation.

To elucidate the structural basis of NICOL’s role in ROS1 activation, we determined the cryo-EM structure of the cROS1/cNEL/hNICOL complex (Supplementary Fig. 10). Initial cryo-EM analysis showed that, unlike the flexible state of cNEL in the cROS1/cNEL complex, cNEL forms a rigid asymmetric dimer in the presence of hNICOL, suggesting that hNICOL binding stabilizes the cNEL dimer (Fig. 3b).

Two distinct classes of the cROS1/cNEL/hNICOL complex were identified, each containing a single cROS1 molecule bound to either protomer 1 or protomer 2 of the cNEL dimer via the same VWC2–β1 interface observed in the cROS1/cNEL complex (Fig. 3b, c). The conformation of cROS1 in this complex remains virtually identical to ligand-free cROS1 and cROS1 in the cROS1/cNEL complex, confirming the notion that cNEL does not induce any conformational changes to cROS1. When the models of the two distinct classes are superimposed based on cNEL, the head region of the cROS1 molecule from one structure clashes severely with that from the second structure, explaining why only one cROS1 molecule can engage the asymmetric cNEL dimer at a time (Fig. 3c).

The structure of 2:1 cNEL/hNICOL complex
After applying focused 3D refinement with density subtraction22, the cryo-EM map of the cNEL/hNICOL complex was resolved at 4.1Å resolution, allowing us to build a complete model for this complex (Fig. 3d, e). In this batwing shaped structure, the two cNEL protomers interact primarily through homotypic contacts between their CC domains (Fig. 3d). The cNEL dimer is further stabilized by a domain-swapping mechanism, in which the LamG domain of one protomer engages the other protomer (Fig. 4a, b).

Notably, only a single hNICOL molecule was observed in the cryo-EM map. The middle part of hNICOL adopts an α-helical conformation and forms a three-helix bundle with the CC domains of both cNEL protomers at the center of the batwing shaped complex (Fig. 4a, c). At each end of the hNICOL helix, three inter-molecular disulfide bonds link hNICOL to the CC domains of cNEL protomers 1 and 2, further reinforcing the complex (Fig. 4d). To validate this binding mode, we deleted the CC domain of cNEL, co-expressed and co-purified cNEL-ΔCC mutant with hNICOL, and found that the cNEL-ΔCC mutant no longer binds to hNICOL (Fig. 4e; Supplementary Fig. 11).

In addition to the central hNICOL-helix/cNEL-CC interaction, the VWC5 domain from each cNEL protomer also engages both the hNICOL helix and the CC domain of cNEL. Specifically, the VWC5 domain of protomer 1 interacts with the C-terminal portion of the hNICOL helix and the CC domain of protomer 2, while the VWC5 domain of protomer 2 contacts the N-terminal portion of hNICOL and the CC domain of protomer 1 (Fig. 4c). These asymmetric interactions lead to distinct conformations of the two cNEL protomers (Fig. 3e), resulting in an overall asymmetric dimer configuration. Each cNEL protomer is further stabilized by distinct intra-molecular interactions, predominantly between their EGF and VWC domains.

Within this asymmetric dimer, the LamG domain of each cNEL protomer engages the VWC4 domain of the opposing protomer, albeit in distinct ways (Fig. 4b). On one wing of the asymmetric cNEL/hNICOL complex, a major β-sheet with a concave curvature in the LamG domain of protomer 1 forms tight contacts with the lateral surface of a three-stranded β-sheet in the VWC4 domain of protomer 2 (Fig. 4b). Additionally, a loop between β-strands in this LamG domain makes contacts with the VWC5 domain of protomer 2.

In contrast, on the other wing of the complex, the N-terminal tail of the LamG domain in protomer 2 contacts a surface formed by a β-sheet and a β-hairpin in the VWC4 domain of protomer 1 (Fig. 4b). Furthermore, the N-terminus of hNICOL adopts an extended loop conformation that interacts with both the LamG domain of protomer 2 and the VWC4 domain of protomer 1 (Fig. 4b, c), contributing to the stability of this LamG–VWC4 interface. The LamG domain of protomer 2 also directly interacts with the EGF5 domain of protomer 1 (Fig. 4b), providing an additional stabilizing interaction for the asymmetric cNEL dimer. Notably, because the LamG–VWC4 interactions observed in the cNEL/hNICOL complex do not involve hNICOL, we suspect that the same interactions may also occur in the NICOL-free cNEL dimer.

Proposed activation mechanism of ROS1 induced by NEL and NICOL
As described above, the 2:1 cNEL/hNICOL complex can recruit only a single cROS1 molecule, due to a steric clash that would occur if both protomers simultaneously bound cROS1. Since RTK activation generally requires receptor dimerization or higher-order oligomerization, this 1:2:1 cROS1/cNEL/hNICOL configuration alone cannot explain how NEL and NICOL mediate ROS1 activation. This structural observation suggest that the 1:2:1 complex must engage in higher-order assembly to enable cROS1 activation. Indeed, both SEC-MALS and blue native PAGE (BN-PAGE) results showed that cROS1 alone predominantly exists as a monomer, whereas the cNEL, cNEL/hNICOL, cROS1/cNEL, and cROS1/cNEL/hNICOL complexes can further assemble into higher-order oligomers (Fig. 5a, b).

Supporting this idea, low-pass-filtered cryo-EM maps of both classes of the cROS1/cNEL/hNICOL complex reveal additional density adjacent to the VWC4 domain of protomer 2 that is consistent in size and shape with a LamG domain, suggesting the presence of a second hetero-tetrameric complex (Supplementary Fig. 12). Based on these observations, we propose that a 1:2:1 cROS1/cNEL/hNICOL complex may associate with a second heterotetrametric complex through an interaction between the VWC4 domain from the first complex and the LamG domain from the second complex (Supplementary Fig. 12). Through the same interaction, the cROS1/cNEL/hNICOL complex could form daisy chain-like higher-order oligomers, in which multiple ROS1 molecules can promote their trans-autophosphorylation and activation.

To test this model, we expressed and purified a LamG-deleted version of cNEL, designated cNEL-ΔLamG. SDS-PAGE and pull-down assays demonstrated that cNEL-ΔLamG could still associate with hNICOL (Fig. 4e and Supplementary Fig. 11) and bind cROS1 with the same affinity as the wild-type cNEL/hNICOL complex (Supplementary Fig. 13a). We next assessed whether cNEL/hNICOL can self-assemble into higher-order oligomers using BN-PAGE. Consistent with our structural model, the wild-type cNEL/hNICOL complex formed tetramers, hexamers, and larger oligomeric species, whereas the cNEL-ΔLamG/hNICOL complex failed to form higher-order assemblies (Fig. 5b). Additionally, the cNEL-ΔCC mutant predominantly existed in a monomeric state, with only a small portion forming high-order oligomer (Fig. 5b). In cell-based assays, the wild-type cNEL/hNICOL complex robustly activated cROS1, whereas both the cNEL-ΔLamG/hNICOL and cNEL-ΔCC mutants exhibited remarkably reduced ability on inducing cROS1 activation (Fig. 5c). Furthermore, we titrated a cNEL mutant lacking both the Coiled-Coil and LamG domains (cNEL-ΔCC-ΔLamG), which contains the VWC2 domain required for strong cROS1 binding but cannot induce cROS1 activation (Supplementary Fig. 13b), into wild-type cNEL/hNICOL-dependent cROS1 activation assays. The results showed that cNEL-ΔCC-ΔLamG inhibited WT cNEL/hNICOL–induced cROS1 activation in a dose-dependent manner (Supplementary Fig. 13c). These findings suggest that both the binding of cNEL/hNICOL to cROS1 and the ligand induced higher-order oligomerization are required for cROS1 activation.

Methods
Oligonucleotides and plasmids
Detailed oligonucleotides and plasmid information is provided within the Source Data File. All of the primers were synthesized by Sigma Aldrich. The cDNA templates for cROS1 and cNEL were purchased from GenScript. The ORF sequences for cNICOL and hNICOL amplification were synthesized by IDT. All the constructs were made according to the directions described in “Method” section.

Chicken ROS1 expression and purification
The DNA sequence encoding the full-length extracellular domain of cROS1 (residues 1–1873) was amplified by polymerase chain reaction (PCR) from a cDNA template (GenScript, Oga00025) and cloned into our previously modified pEZT-BM vector, which contains an H3C cleavage site followed by Tsi and His tags at the C-terminus, using the Gibson assembly method. The amino-acid sequences of linkers/H3C cleavage site/tags appended to the C-terminus is “GTSSG-LEVLFQGP-VDFDKTLTHPNGLVVERPVGFDARRSAEGFRFDEGGKLRNPRQLEVQRQDAPPPPDLASRRLGDGEARYKVEEDDGGSAGSEYRLWAAKPAGARWIVVSASEQSEDGEPTFALAWALLERARLQ-SS-HHHHHHHH”.

The expression and purification of cROS1 were performed as described in previous study with some modifications30. In brief, pEZT-BM-cROS1-H3C-Tsi-His8 plasmid was transformed into the DH10Bac bacteria to generate bacmid DNA. The bacmid DNA was introduced into Sf9 (Gibco, 11496015) cell cultures in 6-well culture plates (1 million per well) with 2 ml Insect-XPRESS™ Medium (Lonza, 12730Q) at 27°C to produce recombinant baculovirus by using Cell-fectin reagent (20 μg bacmid DNA: 8 μL Cellfectin™ II Reagent (10362100)). Twelve hours after transfection, 1% antibiotics and 2% FBS were added. Six days after transfection, collect cell culture medium containing P1 virus. P2 viruses were produced by adding 0.5 ml P1 containing medium into 50 ml Sf9 cell culture (cell density around 2.5 million per ml). Five days after infection, collect cell culture medium containing P2 virus. 3 ml of P2 virus containing medium add into 300 ml Sf9 cells culture to produce P3 virus. Four days after P2 infection, collect P3 virus to express target proteins.

Protein was expressed in FreeStyle 293-F (Invitrogen R79007) cells cultured in FreeStyle™ 293 Expression Medium (Gibco, 12338-018) by infecting the virus at a ratio of 3:100 (virus: cell, v/v). The infected cells were supplemented with 10 mM sodium butyrate to boost protein expression 12 h after virus infection. The cells were cultured at 37°C and 8% CO2.

The cell culture medium was harvested 96 h after sodium butyrate supplement and filtered. CaCl2 was added to the filtered cROS1-containing cell culture medium to a final concentration of 5 mM prior to incubation with pre-equilibrated Ni²⁺-NTA (nickel) resin (Cubo Biotech, 75105) at 4°C for 6–8 h. The cell culture medium, together with resin, was loaded into a column and drained under gravity. The nickel resin was washed with washing buffer 1 (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 3 mM CaCl2, 20 mM imidazole) by gravity flow for 2 times and then washed with washed with wash buffer 2 (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 3 mM CaCl2, 40 mM imidazole). The resin was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 250 mM imidazole). The eluted protein was concentrated and cleavage by HRV-3C protease to remove the tag. The protein sample was then run on Superose 6 increase 10/300 GL size-exclusion column with GF buffer (20 mM HEPES-Na buffer pH 7.5; 150 mM NaCl and 2 mM CaCl2). The peak fractions were combined, concentrated, flash frozen with liquid nitrogen, and then stored at −80°C for subsequent experiments.

Chicken NEL expression and purification
DNA sequence encoding the full-length of cNEL was amplified by Polymerase Chain Reaction (PCR) from cDNA sequence (GenScript, Oga27602) and cloned into our previously modified pEZT-BM vector containing a H3C cleavage site followed by Tsi and His tag on C-terminal. Mutant cNEL-expressing plasmids were generated from the pEZT-BM-cNEL-H3C-Tsi-His8 vector using Q5 site-directed mutagenesis. For cNEL-ΔLamG mutant, the sequence before CC domain (28-212) was deleted. The deleted sequence is LQIDVLSELGLPGYAAGVRQVPGLHNGSKAFLFPDTSRSVKASPETAEIFFQKLRNKYEFTILVTLKQAHLNSGVIFSIHHLDHRYLELESSGHRNEIRLHYRTGSHRSHTEVFPYILADDKWHRLSLAISASHLILHVDCNKIYERVVEKPFMDLPVGTTFWLGQRNNAHGYFKGIMQDVQLLV. For cNEL-ΔCC mutant, the sequence around CC domain (228-270) CPTCNDFHGLVQKIMELQDILAKTSAKLSQAEQRMNKLDQCYC was replaced by linker sequence SPTSGSGSSGGSSGGSSGGSSGGSDQSYS.

For cNEL-ΔCC-ΔLamG mutant, the sequence around CC domain and before CC domain (28-270) was deleted. The deleted sequence is LQIDVLSELGLPGYAAGVRQVPGLHNGSKAFLFPDTSRSVKASPETAEIFFQKLRNKYEFTILVTLKQAHLNSGVIFSIHHLDHRYLELESSGHRNEIRLHYRTGSHRSHTEVFPYILADDKWHRLSLAISASHLILHVDCNKIYERVVEKPFMDLPVGTTFWLGQRNNAHGYFKGIMQDVQLLVMPQGFISQCPDLNRTCPTCNDFHGLVQKIMELQDILAKTSAKLSQAEQRMNKLDQCYC.

The baculovirus production, protein expression, and purification procedures for cNEL were the same as those used for cROS1, as described above.

Co-expression and co-purification of cNEL/hNICOL complex
DNA sequence encoding human NICOL and chicken NICOL were synthesized by Integrated DNA Technologies (IDT, Inc.), and cloned into our previously modified pEZT-BM with Gibson assembly method to construct pEZT-BM-hNICOL-H3C-Tsi-His8 expression vector.

The baculovirus production for cNEL and hNICOL was the same as that used for cROS1, as described above. To isolate cNEL/hNICOL protein complex, we applied co-expression approach by adding cNEL and hNICOL expression baculovirus into Freestyle 293-F cells at the same time. The cNEL and hNICOL viruses were added at a ratio of 1:2 (20 ml cNEL-encoding P3 virus and 40 ml hNICOL-encoding P3 virus were added to 1 L Freestyle 293-F cell culture) to ensure excess expression of hNICOL. The following procedures for expression and purification of cNEL/hNICOL were totally the same as those used for cROS1.

cROS1/cNEL or cROS1/cNEL/hNICOL complex reconstitution
Gel filtration purified cNEL or cNEL/hNICOL was mixed with cROS1 at molar ratio of 1:1 and incubated on ice for 30 min to reconstitute ligand-receptor complex. The sample was then run on a Superose 6 increase 10/300 GL size-exclusion column with GF buffer (20 mM HEPES-Na buffer pH 7.5; 150 mM NaCl and 2 mM CaCl2). The peak fractions were collected and concentrated using an Amicon Ultra concentrator with a 100 kDa cut-off (Millipore) to different concentration accordingly.

ROS1 signaling assay
For ROS1 signaling assay, the DNA sequence encoding the full-length of cROS1 was amplified by polymerase chain reaction (PCR) from a cDNA template (GenScript, Oga00025) and cloned into modified pcDNA3.1(-) vector, which contains an EGFP tag followed by a myc tag at the C-terminus, using the Gibson assembly method. Point mutant ROS1-expressing plasmids were generated from the pcDNA3.1-cROS1-EGFP-myc vector using Q5 site-directed mutagenesis.

For phosphorylation of ERK assessment, Vigorous 293FT (Invitrogen, R70007) cells were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics under standard cell culture conditions. Subculture the cells into 6-well culture plates and seed approximately 0.8 million cells per well when they reach 80% confluency. After 1 day, plasmid transfection was performed using PEI at a 1:3 (DNA: PEI) mass ratio to express EGFP-Myc-tagged cROS1 and its mutants in 293FT cells. One day after plasmid transfection, most cells exhibited green fluorescence. The cells were then serum-starved for 10 h. Serum-starved cells were treated for 25 minutes with cNEL, cNEL/hNICOL, or mutant ligands, each diluted in high-glucose DMEM without serum to a final concentration of 100 nM. After treatment, cells were incubated with cell lysis buffer [20 mM Hepes 7.5; 150 mM NaCl; 1% triton X100] supplemented with cOmplete Protease Inhibitor Cocktail (Roche) on ice for 10 min. After centrifugation at 20,600 g at 4°C for 15 min, cell lysate supernatant samples were made with 4X SDS DTT containing buffer. The samples were then separated by SDS-PAGE and analyzed by Western blotting. anti-Myc (1:2000; Cell Signaling, 2278), anti-pERK1/2 (1:2000; Cell Signaling, 4370), anti-ERK1/2 (1:2000; Cell Signaling, 4695), and anti-alpha-tubulin (1:2000; Cell Signaling, 3873) were used as primary antibodies. Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling, 7074) and Anti-mouse IgG, HRP-linked Antibody (Cell Signaling, 7076) were used as secondary antibodies accordingly.

For phosphorylation assessment of cROS1, NCI-H1299 (ATCC, CRL-5803™) cells were cultured in RPMI-1640 medium (Gibco, 11875093) supplemented with 10% FBS and 1% antibiotics under standard cell culture conditions. Cells were subcultured into 6-well plates and seeded at approximately 1×10⁶ cells per well. After 2 days, when the cells reached ~80–87% confluency, plasmid transfection was performed using Lipofectamine™ 2000 to express EGFP-Myc-tagged cROS1. 12 h after transfection, the medium was replaced with fresh growth medium. Two days after transfection, the cells were serum-starved for 16 h. Serum-starved cells were then treated with cNEL/hNICOL (100 nM, diluted in serum-free RPMI-1640) for 25 min. Subsequent cell lysis and western blot procedures were performed as described above for 293FT cells. Anti-pROS1 (Tyr2274) (1:1000; Cell Signaling, 3078) was used as primary antibodies.

Statistics and reproducibility
ImageJ 1.53e was used to quantify band intensities from Western blot experiments. GraphPad Prism 5 was used to generate graphs and perform statistical analyses. Data are presented as mean ± s.d. from n=3 independent experiments. A two-tailed Student’s t-test was used for comparisons between the ligand-site–mutated group and the no-ligand–treated group. No statistical method was used to predetermine sample size. No data were excluded from the analyses. Randomization and blinding methods were not used.

Pull-down assay
For samples involving cROS1/cNEL interface mutants, 10 µg of bait protein (cNEL-TSI3-His or TSI tagged mutants) and 10 µg of prey protein (cROS1) were mixed in 500 µl of TBS buffer (20 mM Tris-Cl, pH 7.5; 150 mM NaCl; 0.1% Tween-20) supplemented with 2 mM CaCl₂, and incubated on ice for 1 h. Pre-equilibrated TSE resin was then added, and the mixture was incubated overnight at 4°C with gentle rotation. After incubation, the samples were centrifuged at 400 × g for 2 min, and the supernatant was discarded. The resin was washed three times with TBS buffer containing 2 mM CaCl₂ to remove unbound proteins. Bound proteins were eluted by boiling the resin in SDS loading buffer supplemented with DTT, and analyzed by SDS–PAGE.

Native gel analyses cNEL/hNICOL oligomerization
Native gel electrophoresis was performed as previously described by our laboratory31. Briefly, purified cNEL/hNICOL wild-type or mutant protein complexes (15 µg) were prepared in GF buffer (20 mM HEPES-Na, pH 7.5; 150 mM NaCl; 2 mM CaCl₂), mixed with NativePAGE™ sample buffer (Invitrogen, Cat. No. BN20032), and resolved on a 3–12% Bis-Tris gradient native gel (Invitrogen, Cat. No. BN2012BX10).

SEC-MALS experiments
All size-exclusion chromatographic multi-angle light-scattering (SEC-MALS) experiments were conducted at 4°C. A Superdex 200 Increase GL 10/300 column, attached to an ÄKTA pure system (GE), was equilibrated with MALS buffer (20 mM HEPES, pH 7.5 150 mM NaCl, and 2 mM CaCl2). Protein samples (200 μL; filtered through a 0.22 μm centrifugal filter) at 1 mg/mL were injected onto the column and detected with the ÄKTA’s UV detector and a miniDAWN TREOS detector (Wyatt Technologies). The flow rate was 0.5 mL/min. Data were analyzed using ASTRA version 7.1.0.29 (Wyatt). Concentration information was gleaned from the UV absorbance and the respective calculated mass-based extinction coefficients. For protein complexes, these values were surmised based on relative elution volumes and likely molar ratios. A sample of bovine serum albumen (Pierce) was used to calibrate the apparatus. Reported molar masses are the weight-averages of the masses observed across the dominant light-scattering peak. In some cases, it was necessary to exclude strongly sloping and/or noisy parts of the molar-mass distribution in the calculation of these weight-averages.

BLI assay
Binding affinity of ROS1 to NEL or NEL/NICOL was determined by BLI using the Octet® R2 system (Sartorius). The cROS1 protein was biotinylated using EZ-Link NHS-PEG4-Biotin (ThermoFisher Scientific, MAN0016360) at a protein-to-biotin molar ratio of 1:1. Excess biotin was removed by gel filtration. 5 nM of biotinylated cROS1 in kinetic buffer (20 mM Hepes, 150 mM NaCl, and 2 mM CaCl2 at pH 7.5, 0.1% (w/v) BSA, and 0.02% Tween-20) were captured on Streptavidin (SA) Biosensors (Sartorius, 18-0009) for 120 s, followed by a baseline measurement in kinetic buffer for 180 s. cNEL/hNICOL (containing 3.13 nM, 6.25 nM, 12.5 nM, 25 nM and 50 nM) or cNEL (containing 0.41 nM, 1.23 nM, 3.7 nM, 11.1 nm and 33.3 nM) were associated to biotinylated ROS1-coated sensor-tip surfaces (180 s), followed by a dissociation phase (240 s). To account for potential non-specific interactions, we dipped sensors lacking biotinylated cROS1 into cNEL or cNEL/hNICOL solutions, and to account for dissociation of biotinylated cROS1 from the sensor over the course of the experiment, we dipped the biotinylated cROS1-coated sensor into kinetic buffer lacking analyte followed by subtraction of the signals of both control measurements using the Octet® Analysis Studio software.

Cryo-EM grids preparation
Quantifoil R1.2/1.3 300-mesh gold holey carbon grids (Quantifoil, Micro Tools GmbH, Germany) were glow-discharged for 60 s with PELCO easiGlow™ Glow Discharge Cleaning System. Cryo-EM grids were prepared with a Vitrobot Mark IV (FEI). For cROS1/cNEL sample, the cryo-EM grid was prepared by applying 3.8 μl of the sample at the concentration of 0.8 mg/ml to glow-discharged grids. Grids were blotted for 3.0 s under 100% humidity at 4°C before being plunged into nitrogen-cooled liquid ethane using the Mark IV Vitrobot. For cROS1 only sample or cROS1/cNEL/hNICOL sample, the samples were concentrated to 8 mg/ml, and 3 mM fluorinated Fos-Choline-8 (Anatrace) (final concentration) was added to the concentrated sample immediately before preparing the cryo-EM grids. The cryo-EM grids were prepared by applying 3.8 μl of the protein samples to glow-discharged Quantifoil R1.2/1.3 300-mesh gold holey carbon grids. Grids were blotted for 4.0 s under 100% humidity at 4°C before being plunged into the liquid ethane using the Mark IV Vitrobot.

Cryo-EM data collection and image processing
EM data acquisition, image processing, and model building, and refinement were performed following previous protocols with some modifications. Micrographs were collected in the counting mode on Titan Krios microscopes (ThermoFisher Scientific) with K3 Summit direct electron detectors (Gatan). The nominal magnification and pixel size of each dataset are summarized in Table 1. Motion-correction and dose-weighting of the micrographs were carried out using the Motioncor2 program (version 1.2)32. GCTF 1.06 was used for CTF correction33. Template-based particle picking was carried out using the autopick tool in RELION 5.034. Particles were binned with 4 times and cleaned up with multiple rounds of 2D and 3D classification in RELION. The exact procedures are summarized in Supplementary Figs. The initial mode for 3D classification and refinement was generated using the SGD method in RELION. The final refinement was carried out with unbinned particles, and the refined maps were further improved by using Bayesian polishing, blush regularization, and CTF refinement at the final stage. The Fourier Shell Correlation (FSC) 0.143 criterion was used for estimating the resolution of the maps. Local resolution was calculated in RELION.

For the dataset of ligand-free-cROS1, the 3D classification with small angle sampling search revealed three overall conformations (3.7-degree sampling, 20 degrees searching range), with different relative angles between the “head” and “leg” regions of cROS1. After focused refinement using a soft mask around either the “head” or “leg” region, the cryo-EM densities for both the “head” and “leg” regions were improved significantly and were resolved at 2.9Å and 3.6Å resolution, respectively.

For the dataset of cROS1/cNEL complex, the initial cryo-EM map of cROS1/cNEL was resolved at 2.7Å resolution, but only the VWC2 domain of cNEL was resolved. After focused classifications using a soft mask on the top region of the cROS1/cNEL complex, the EGF1, EGF2, VWC1, and VWC2 domains were resolved. The overall resolution for this map is 3.8Å.

For the dataset of cROS1/cNEL/hNICOL, the part corresponding to the cNEL/hNICOL was poorly resolved in the initial cryo-EM determination. After focused classification with density subtraction22, the cryo-EM density of 2:1 cNEL/hNICOL complex was improved remarkably. Before the focused refinement of the 2:1 cNEL/hNICOL complex, we centered the density corresponding to the 2:1 cNEL/hNICOL complex to the center of the box and re-extracted all the particles to a smaller box. The focused 3D classification was performed using a smaller angular sampling (3.7 degree) and local search (20 degree). Two different conformations were identified, with one ROS1 molecule bound with each of the cNEL protomers, respectively. The 3D refinement of original particles without density subtraction resulted in cryo-EM maps for two distinct conformations of 1:2:1 cROS/cNEL/hNICOL complex, at the resolution of 4Å and 4.2Å, respectively.

Model building and refinement
Model building was initiated by docking of the AlphaFold predicted models of the individual proteins into the cryo-EM maps. The models were adjusted manually and refined using the ISOLDE plugin in ChimeraX35,36. N-glycans were added to the models based on the density using ISOLDE. The coordinates and atomic b-factors of the models were further refined using the real-space refinement module in Phenix37, with secondary structure and Ramachandran restraints. The validation of the models was carried out using the Molprobity module in Phenix.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an interesting and timely study that advances our understanding of vertebrate (chicken) ROS1 regulation. To date, only Drosophila ROS1 (Sevenless) has been structurally and mechanistically characterized, and it employs an entirely different ligand and activation mechanism that is not conserved in vertebrates. Here, the authors use chicken ROS1 (cROS1) and its ligand NEL, with or without the co-ligand NICOL, to determine structures that point towards a novel mode of regulation. The cryo-EM work appears to be of high quality and at resolutions sufficient to define observed interactions. These findings represent an important step toward understanding the molecular basis of vertebrate ROS1 signaling. However, several major issues need to be addressed before the conclusions can be fully supported.\n\nMajor Concerns\n1. Supporting biophysical characterization\no While the structures are well executed, there is no quantitative binding analysis (SPR/BLI) comparing ROS1–NEL and ROS1–NEL/NICOL interactions.\no No analytical ultracentrifugation (AUC) or SEC-MALS data are provided to define the oligomeric states of the complexes in solution. In particular, SEC-MALS comparisons of ROS1 alone, ROS1–NEL, and ROS1–NEL/NICOL would clarify stoichiometry and complex formation.\no Even SEC traces for ROS1–NEL/NICOL versus ROS1–NEL are absent.\n2. Cell-based activation studies\no ROS1 phosphorylation is not measured; only downstream ERK phosphorylation is reported. In a transient expression system, direct evidence of ROS1 activation is essential.\no The fact that point mutants and truncations still show elevated pERK relative to the no-ligand control is puzzling and warrants explanation in the context of proposed mechanism.\no An inhibitory experiment could strengthen the claims: titrating mutant or truncated NEL/NICOL into wild-type ROS1 activation assays to assess suppression of signal.\n3. Limited kinetic and dose–response analysis\no Activation assays are performed at a single high concentration (100 nM) and a single long time point (25 min). This design precludes understanding the relative kinetics of activation by NEL versus NEL/NICOL.\no A time course and dose–response analysis could reveal whether NEL/NICOL induces prolonged activation versus NEL inducing transient but robust activation.\n4. Unresolved questions about NEL oligomeric state\no The manuscript does not address whether NEL alone is trimeric, and the observation that NEL alone can activate ROS1 appears inconsistent with an absolute requirement for NICOL and subsequent Laminin G directed polymerization. (If all laminin G domains are engaged in the NEL–ROS1 interface, how could NEL still facilitate higher-order clustering? The structural rationale is unclear.)\n5. Structural modeling and interpretation\no Discussions of laminin G interactions are dense and difficult to follow without clearer figures.\no The low-resolution density attributed to higher-order assembly via “blob docking” is not convincing.\no The authors should consider modeling potential dimer-on-dimer arrangements and estimate the resulting kinase domain separations to assess feasibility of activation.\no Why is the cNEL-ROS1 structure the only structure without the leg modeled? The description states that cNEL binding doesn’t cause any changes in cROS1, but this seems to be a potential important change.\no The density of the second bat-wing of cNEL is less well defined than the protomer that shows clear binding to cROS1. Is it possible that a flexible ROS1 could be bound at the second site? This would be similar to the proposal of lamininG induced clustering (which the authors claim can not be seen directly in the cryoEM because of flexibility) and achieve the same signaling.\n6. Ligand species mismatch\no The use of human NICOL with chicken NEL and chicken ROS1 may be problematic. As seen in the supplied alignments, human NICOL contains an additional cysteine that could significantly alter association and activity. The authors should test chicken NICOL in NEL complex to rule out unintended artifacts.\nMinor Comments\n• The title should specify “chicken ROS1” or “avian ROS1” to avoid overgeneralizing.\n• Replace “apo” with “unliganded” or “ligand-free,” as “apo” is typically used for small-molecule–free enzyme forms.\n• Maintain consistent terminology for the unliganded receptor conformation (“extended” vs. “arc-shaped”).\n• The phrase “hNICOL promotes cNEL dimerization” may be misleading, as it implies cNEL is otherwise monomeric. Clarify whether cNEL is trimeric, as expected for other NELLs, and whether NICOL prevents trimerization.\n• Correct typographical errors: e.g., “cNET/hNICOL” should read “cNEL/hNICOL.”\nOverall, the structural data are of high potential interest, but the lack of supporting biochemical and cell-based validation makes it difficult to assess the physiological relevance of the proposed mechanism. Adding quantitative binding studies, oligomeric state analyses, direct ROS1 phosphorylation data, and expanded activation kinetics would substantially strengthen the manuscript.", "reviewer_2": "The authors present the first full-length extracellular domain structure of a higher vertebrate receptor tyrosine kinase (RTK) ROS1 in it’s apo form and bound to the ligand NEL, and a ligand/co-ligand complex NEL/NICOL. This is an exciting finding that provides new insight into oligomerization via interaction with this co-liganded complex. Activation of RTKs, which are important regulatory signaling molecules and common therapeutic targets, is a topic of great current interest as we are beginning to understand that RTK signaling often has several layers of complexity, and this paper provides some of the first direct structural evidence for clustering mediated by co-ligands, which I think will be of great interest to your readership and something that is widely discussed and cited going forward.\nThe authors provide a careful analysis of the three structures and follow up key findings with functional assays. It’s also interesting that sugars are clear mediators of some domain interactions in apo ROS1, highlighting the importance of determining these structures with glycans included, as the authors did here. This is worth mentioning, because historically many RTK structures are deglycosylated, a practice which has clearly hidden important features of structural organization.\nI’ve included a few comments below that I thought were important to make:\nThe authors comment about missing domains from cNEL in the cROS1/cNEL complex, but not on the missing “leg” of the ECD domain of cROS1. Was this also likely due to conformational flexibility, as informed by analysis of 2D classes, or another possible reason? They indicate in the discussion that ROS1 does not undergo signficiant conformational changes upon NEL binding, but it’s unclear that this statement can be made without the entire ECD. Increased head to leg flexibility, for example, could be significant and functionally important. While I don’t think this structure needs to have the leg domain (and it was likely challenging), it would be nice to see some comments and thoughts informed by their experimental data on what may be happening here, especially as leg domain movement would likely be important for bringing intracellular domains in close proximity in oligomers. Of note, more of this is visualized in the co-liganded complex, which I think suggests that the ligands may have different effects on full-length ROS1.\nWhile the authors provided pdb validation reports that for the most part look excellent (90ZH and 90ZC appear to have some validation issues, but this is not uncommon for lower resolution structures), they did not provide any raw data (pdb and generated maps)? It would be best for reviewers to see this data before being sure about their review, as it would be helpful to determine what may have been the source of issues in the validation report (was it truly a function of the resolution or other potential issues). Obviously, due to size we cannot generally share all EM raw data, but the final maps and pdbs are easy to share.\nIn the same note, I assume the residues substituted for functional analysis (i.e., F318A, E541A, F527A and Y553A) were surface exposed an unlikely to impact the tightly bent structure of the ROS1 “head”, but I cannot determine this without the pdbs. If they are not surface exposed or suspected effects on packing could occur, it would suggest impacts to activation could be due to local instability rather than disrupted interactions with ligand. Figure 2D does show these residues, but it’s impossible to use this image for this type of analysis. For example, E541 looks like it has a potential ionic interaction with R368 also of ROS1 such that mutating this to alanine may cause local instability. As such, a clear interpretation that these substitutions impacted interaction with ligand and not folding would be difficult to make. However, given that they mutated several sites independently, it seems likely that ligand interaction is being impacted, making this a less critical point.\nThe color choices for the NEL/NICOL complex (especially in Figure 4) make it quite hard to distinguish the two, as there could be more contrast.\nThe authors argue that liganded ROS1 likely forms higher order oligomers, or arrays, which has been suggested in the activation of other RTKs and is a current hot topic of interest in the field (making this paper highly relevant), but they do not model this in their working model (Figure 6). I think this is a big oversight, as someone reading this figure would just think this represents a model for simple ligand-induced dimerization. Figure 6 would also be stronger as a standalone figure if the complex components were labeled or if there was some sort of legend.\nLastly, I wonder if the authors tried modeling the dimer using the additional LamG density as a start and then tried to create templates from that to see if it was found in the dataset? The additional density does seem to suggest dimers were present in the sample, but at a lower sampling rate. Given that the monomeric complex structure seems pretty rigid, this may be worth trying if they haven’t already. It may provide a bit more information about the receptor:ligand/co-ligand ‘dimer’.", "reviewer_3": "Overall Assessment\nThis manuscript reports the cryo-EM structure of the NEL–NICOL–ROS1 complex, providing high-resolution structural insight into ROS1 activation by its recently identified endogenous ligands, NELL2 and NICOL. While ROS1 has long been recognized in oncology as a receptor tyrosine kinase activated by oncogenic fusion proteins, the physiological ligands and activation mechanisms have remained unclear until recent work identified the NELL2/NICOL complex as a key mediator of testis-to-epididymis signaling and male fertility. From a structural biology perspective, no detailed architecture of this ternary complex has been described, making the present work timely and potentially significant.\nThe study’s key strength lies in the novelty of the structural findings, which extend our understanding of ROS1 signaling beyond the oncogenic context and provide a foundation for exploring distinctive modes of RTK activation. The cryo-EM data reveal a ligand–co-ligand–receptor arrangement that offers a structural framework likely to inspire new hypotheses about receptor clustering and signal initiation. The topic will interest structural biologists, reproductive biologists, and the broader RTK signaling community.\nSeveral aspects would benefit from further attention to maximize impact and clarity. The methodological description—particularly sample preparation and the selection/heterogeneity of conformational states for cryo-EM analysis—is not yet sufficiently detailed to fully assess reproducibility or the basis of structural interpretation. The linkage to prior biochemical/structural literature could be sharpened; the novelty would be even clearer if the authors positioned their results explicitly relative to earlier models and explained methodological differences that may account for discrepancies. In addition, some mechanistic claims, especially regarding ROS1 activation, would benefit from being phrased with greater caution to strengthen their persuasiveness.\nIn summary, this is a potentially high-impact and original study. By adding methodological detail, more explicitly situating the findings within prior literature, and tempering mechanistic proposals where direct evidence is not yet available, the authors can enhance rigor, reproducibility, and interpretability. Addressing the points below should help ensure the work’s significance is fully appreciated by a broad readership.\nNote on referencing: As the submitted PDF lacks line numbering, specific locations are indicated using figure/panel numbers, section headings, and short quoted phrases rather than page/line numbers.\nMajor Comments\nNICOL-free cNEL\n1. According to Extended Data Fig. 1, cROS and cNEL were purified by Ni-affinity chromatography followed by gel filtration on a Superose 6 Increase 10/300 GL column, with peak fractions collected and concentrated. Please indicate whether the gel-filtration step may enrich particular oligomeric states, as this could influence interpretation of subsequent structural analyses.\n2. For recombinant NICOL-free cNEL, please clarify whether it exists primarily as a monomer or dimer. Under non-reducing SDS-PAGE it appears not to be monomeric, and Extended Data Fig. 1 suggests a major disulfide-linked dimer plus some monomer. Stating this explicitly in the Results would aid interpretation of complex-formation data.\n3. While the data indicate that NICOL-free cNEL (~240 kDa) is not a monomer, SDS-PAGE alone may not be sufficient to conclude it is a dimer. If feasible, please consider confirming the oligomeric state by mass spectrometry or an equivalent technique to strengthen the conclusion.\n4. If NICOL-free cNEL does form a dimer, please clarify whether the LamG–VWC4 interaction occurs between protomers. Indicating whether this is clearly observed, unclear, or unresolved would help readers assess the mechanism.\ncNEL–cROS interaction\n5. Extended Data Fig. 1 shows that NICOL-free cNEL used for complex formation with ROS1 was disulfide-linked dimeric rather than monomeric. Explicitly stating this in the Results would contextualize complex formation.\n6. For the cROS–cNEL complex, purified proteins were mixed, separated by gel filtration, and peak fractions were analyzed (Extended Data Fig. 1). Were the SDS-PAGE analyses performed under reducing conditions? If so, what is the band just below cROS—could it be dimeric cNEL or a partially reduced form? Clarifying the analysis conditions and band assignments will aid interpretation.\n7. In the cryo-EM analysis, only the cNEL molecules adjacent to cROS are visible. It remains possible that a cNEL dimer could bind two cROS molecules, particularly if cNEL is more flexible in the absence of NICOL. A brief discussion of this alternative would help frame the observations.\n8. The gel-filtration profile for cROS–cNEL shows an additional early-eluting peak around column volume ~8. Could this correspond to a cNEL dimer bound to two cROS molecules? Providing an interpretation would be informative for readers considering alternative stoichiometries.\ncNEL–hNICOL interaction\n9. Please discuss, even briefly, structural differences between previously reported NELL2/NICOL complexes and the complex proposed here. Indicating whether differences are methodological, biologically relevant, or both would help contextualize the novelty.\n10. Were there prior predictions or preliminary data suggesting that NELL2 and NICOL might form a covalent complex? If so, summarizing them would provide helpful context.\n11. For the statement, “Consistent with previous findings, our cell-based assays demonstrated that the cNEL/hNICOL complex activates ROS1 much more robustly than cNEL alone,” please verify that the supporting citations are accurate and that the prior findings are represented precisely.\ncNEL–hNICOL–cROS interaction\n12. Prior studies reported NELL2 as a trimer, with no evidence of a covalent NELL2–NICOL complex. The finding here of a covalent 2:1 NELL2–NICOL complex is highly novel. Explicitly citing the prior work and highlighting the differences would underscore originality.\n13. The observation that the NELL2–NICOL complex has two ROS1-binding sites, yet no complex with both sites occupied was observed, may relate to sample preparation—specifically, collecting fractions containing only singly bound complexes (Extended Data Fig. 1). Acknowledging this possibility would temper conclusions about stoichiometry.\n14. The manuscript states that cNEL is “flexible and monomeric” in the cROS–cNEL complex but “rigid and dimeric” in the presence of hNICOL. Given that the cNEL used for cROS–cNEL complex formation was already dimeric, please reconcile these statements to ensure internal consistency.\n15. The steric-clash model preventing simultaneous binding of two ROS1 molecules to a cNEL dimer is interesting. Please specify the extent/nature of the predicted overlap to help readers assess the basis of this conclusion.\nROS1 activation mechanism\n16. The proposed activation model involving higher-order multimerization via VWC4–LamG interactions is intriguing but remains speculative based on the current data. Consider moving this to the Discussion, with appropriate caveats, and note explicitly that direct structural evidence is not yet available.\n17. Please clarify how cNEL and cNEL–hNICOL mutants used in BN-PAGE and cell-based assays were prepared. In particular, indicate whether gel filtration was used to isolate fractions of defined oligomeric state, which is essential for evaluating the activation mechanism.\n18. The finding that cNEL–ΔLamG–hNICOL fails to form multimers in BN-PAGE and cannot activate ROS1 despite binding it is mechanistically important. Emphasizing this in the Results and linking it clearly to the proposed model would be helpful.\n19. In the section where the authors state that “the cNEL–ΔCC mutant predominantly existed in a monomeric state,” yet BN-PAGE shows multimers: if only monomeric fractions were used for the cell-based assay, this could explain the lack of activation. Clarification would aid interpretation.\n20. Since NICOL-free cNEL can form dimers, could it also form higher-order assemblies via VWC4–LamG interactions to activate ROS1? If so, what is the role of hNICOL in this model, particularly with respect to VWC5–NICOL/CC interactions? If unresolved, stating this explicitly would be useful.\n21. The mechanism proposes that multiple ROS1 molecules bind a single ligand assembly and approach one another sufficiently for intermolecular phosphorylation. As this arrangement is not directly visualized, acknowledging this limitation would strengthen the Discussion.\n22. The manuscript contrasts a “classical RTK” model with a distinct ROS1 activation mechanism. This perspective is potentially correct, but conclusions should be tempered to reflect remaining uncertainties and avoid overstatement.\n23. The statement that “ROS1 is, to date, the only known RTK that depends on a co-ligand, NICOL, for activation” is not precise, as FGFR activation by FGF requires heparin/heparan sulfate, which function as co-ligands. Please revise accordingly.\n24. The claim that NICOL “stabilizes cNEL into a rigid conformation optimal for ROS1 activation” could be reconsidered. The data may instead support higher-order oligomerization via LamG–VWC4 interactions as the critical determinant. Clarifying this distinction would avoid overinterpretation.\nMinor Comments\n25. For cROS1-H3C-Tsi-His8, hNICOL-H3C-Tsi-His8, cNEL-H3C-Tsi-His8, cNEL-ΔLamG, and cNEL-ΔCC, please provide the amino-acid sequences of linkers/tags appended to the C-terminus in Methods to facilitate reproducibility.\n26. In the section discussing the Drosophila ROS1 homolog Sevenless (Sev) and its ligand Boss, the content is interesting but not directly tied to the manuscript’s core novelty. In addition, the statement “we did not observe cROS1 dimerization under acidic conditions” lacks supporting data. Consider shortening this section or moving it to Supplementary Information.\n27. In Fig. 2a and Fig. 3e, the numbering/labeling of VWC domains appears incorrect. Please correct for accuracy and clarity."}, "author_response": "We thank the reviewer for the positive assessment of our manuscript.\n\nMajor Concerns\n1. Supporting biophysical characterization\no While the structures are well executed, there is no quantitative binding analysis (SPR/BLI) comparing ROS1–NEL and ROS1–NEL/NICOL interactions.\nResponse: Good point. In response to the reviewer’s comment, we analyzed the binding affinity using BLI. The results showed that ROS1–NEL and ROS1–NEL/NICOL have nearly identical binding affinities, with the K values in the single-digit nanomolar range. These new results have been added to the revised manuscript (new Supplementary Figure 3).\no No analytical ultracentrifugation (AUC) or SEC-MALS data are provided to define the oligomeric states of the complexes in solution. In particular, SEC-MALS comparisons of ROS1 alone, ROS1–NEL, and ROS1–NEL/NICOL would clarify stoichiometry and complex formation.\nResponse: Thanks for the good suggestions. We have performed SEC-MALS characterization on cNEL, cNEL/hNICOL, cROS1, cROS1/cNEL, and cROS1/cNEL/hNICOL sample. The results showed that cROS1 alone is a monomer, but forming large high-order oligomer when bound to either cNEL or cNEL/hNICOL complex. cNEL or cNEL/hNICOL can also self-assemble into a higher-order complex. All these results are consistent with our blue native PAGE results, supporting our proposed activation model. These new results have been included in the new Figure. 5a.\no Even SEC traces for ROS1–NEL/NICOL versus ROS1–NEL are absent.\nResponse: Point accepted. In the revised manuscript, we have included the SEC result of the ROS1–NEL/NICOL complexes and compared its SEC profile with that of ROS1–NEL (new Supplementary Figure 6).\n2. Cell-based activation studies\no ROS1 phosphorylation is not measured; only downstream ERK phosphorylation is reported. In a transient expression system, direct evidence of ROS1 activation is essential.\nResponse: Thank you to the reviewer for raising this important point. We initially examined cROS1 phosphorylation in 293T cells but did not observe any difference between the cNEL/hNICOL-treated and control groups. We then tested ROS1 phosphorylation in H1299 cells, where the results showed that cNEL/hNICOL moderately stimulates cROS1 and ERK phosphorylation (new Supplementary Figure 8). Although the increase in ROS1 phosphorylation was modest, the results were reproducible and statistically significant. Notably, in the original study that identified NELL2 as a ROS1 ligand (PMID: 32499443), the authors reported only ERK phosphorylation rather than ROS1 phosphorylation, suggesting that direct detection of ROS1 phosphorylation upon NELL2 stimulation is technically challenging. We speculate that, although ROS1 autophosphorylation is relatively weak, signaling strength may be amplified through the downstream pathway. Given the weak autophosphorylation of ROS1, we chose to evaluate all mutants based on their effects on ERK phosphorylation in 293T cells.\no The fact that point mutants and truncations still show elevated pERK relative to the no-ligand control is puzzling and warrants explanation in the context of proposed mechanism.\nResponse: Thank you to the reviewer for raising this important point. Our new BLI results showed that cNEL binds to cROS1 with extremely high affinity (K ≈ 2 nM). We suspect that the point mutation alone may not be sufficient to completely disrupt the interaction between cROS1 and cNEL. Furthermore, our native gel results indicated that the cNEL-ΔCC/hNICOL and cNEL-ΔLamG/hNICOL truncations still displayed higher-order oligomerization, albeit at a lower level than the wild type complex (Fig. 5b). These results are consistent with the weakened but not abolished pERK elicited by the point mutants and truncations.\no An inhibitory experiment could strengthen the claims: titrating mutant or truncated NEL/NICOL into wild-type ROS1 activation assays to assess suppression of signal.\nResponse: Thank you to the reviewer for this excellent suggestion. We titrated a NEL mutant lacking both the Coiled-Coil and LamG domains (cNEL-ΔCC-ΔLamG) into wild-type cNEL cROS1 activation assays. Our results showed that cNEL-ΔCC-ΔLamG inhibited WT cNEL–induced cROS1 activation in a dose-dependent manner. This finding further supports that higher-order oligomerization of the cNEL/hNICOL complex, in addition to receptor binding, is required for cROS1 activation. These new results have been included in the revised manuscript (new Supplementary Figure 13c).\n3. Limited kinetic and dose–response analysis\no Activation assays are performed at a single high concentration (100 nM) and a single long time point (25 min). This design precludes understanding the relative kinetics of activation by NEL versus NEL/NICOL.\no A time course and dose–response analysis could reveal whether NEL/NICOL induces prolonged activation versus NEL inducing transient but robust activation.\nResponse: Thank the reviewer for this excellent suggestion. Following the suggestion, we performed cell-based assays using a range of ligand concentrations. The results showed that the activation of ROS1 reaches plateau at around 100 nM concentration of cNEL/hNICOL. In contrast, cNEL cannot robustly induce ROS1 activation even at concentrations up to 2000 nM. These results further demonstrated the critical role of NICOL in NEL induced ROS1 activation (new Supplementary Figure 9a). Furthermore, our time course analysis indicated that the maximal activation of ROS1 by either cNEL/hNICOL complex or cNEL alone was reached at ~30 min (new Supplementary Figure 9b), although the efficacy in ROS1 activation by cNEL alone was much weaker. These data further suggest that the NEL alone is not optimal for ROS1 activation.\n4. Unresolved questions about NEL oligomeric state\no The manuscript does not address whether NEL alone is trimeric, and the observation that NEL alone can activate ROS1 appears inconsistent with an absolute requirement for NICOL and subsequent Laminin G directed polymerization. (If all laminin G domains are engaged in the NEL–ROS1 interface, how could NEL still facilitate higher-order clustering? The structural rationale is unclear.)\nResponse: Good point. Our SDS-PAGE results show that cNEL alone primarily exists as a disulfide-linked dimer and can further oligomerize, capable of activating ROS weakly. We speculate that NICOL may facilitate the formation of higher-order receptor–ligand assemblies that position multiple ROS1 molecules in a more favorable configuration for more potent activation. However, this remains speculative, as such higher-order complexes were not directly observed in our cryo-EM analysis. In addition, our biochemical and cryo-EM data indicate that cNEL exhibits markedly improved biochemical stability and reduced aggregation in the presence of NICOL, suggesting that NICOL may enhance the biological activity of NEL by stabilizing its structure and improving its solubility. Interestingly, NEL and NICOL display highly similar expression patterns across tissues. For example, they are both highly expressed in the brain and male reproductive tissues. This raises the intriguing possibility that NEL is always associated with NICOL in vivo and that NICOL functions as an constitutive structural partner of NEL. We have briefly discussed these points and their limitations in the revised manuscript.\nWe apologize for the confusion regarding the NEL-ROS1 interface. The VWC2 domain of cNEL, not the LamG, is required for cROS1 binding. Based on our truncation results, we propose that the LamG domain is critical for mediating higher-order clustering.\n5. Structural modeling and interpretation\no Discussions of laminin G interactions are dense and difficult to follow without clearer figures.\no The low-resolution density attributed to higher-order assembly via “blob docking” is not convincing.\no The authors should consider modeling potential dimer-on-dimer arrangements and estimate the resulting kinase domain separations to assess feasibility of activation.\nResponse: Thank you to the reviewer for raising this critical point. We agree with the reviewer that the quality of our higher-order ROS1/NEL/NICOL complex structure is insufficient for precise modeling. In the revised manuscript, we have moved Fig. 5a and 5b to the supplementary materials and substantially reduced the discussion regarding how the second 1:2:1 ROS1/NEL/NICOL complex engages the first through LamG-mediated interactions.\nIn the revised manuscript, we have rewritten the section of “Proposed activation mechanism”.\nWe now propose an activation model for ROS1 based on our native gel and SEC-MALS results, which demonstrate that cNEL/hNICOL and cROS1/cNEL/hNICOL can further assemble into higher-order complexes. We briefly note that, based on the size and shape of the additional density, the higher-order assembly may be mediated by LamG interactions, a hypothesis partially supported by our LamG truncation data. Finally, we have added to the discussion in the revised manuscript that further structural studies will be required to elucidate the molecular details underlying the assembly of higher-order ROS1/NEL/NICOL complexes.\no Why is the cNEL-ROS1 structure the only structure without the leg modeled? The description states that cNEL binding doesn’t cause any changes in cROS1, but this seems to be a potential important change.\nResponse: We thank the reviewer for raising this issue. The “leg” region was not resolved in the cryo-EM structure of the cROS1-cNEL complex. We do not believe that cNEL binding increases conformational flexibility between the head and leg regions of cROS1, because: (1) the cNEL binding site on ROS1 is far from the joint between the head and leg regions, and (2) the leg region of ROS1 was resolved in the cryo-EM structure of the cROS1/cNEL/hNICOL complex, showing no structural changes compared to ROS1 alone.\nWe propose an alternative explanation for the poorly resolved ROS1 leg in the cROS1/cNEL complex. Both our BN-PAGE and new SEC-MALS results (Fig. 5) indicate that cNEL alone exhibits poor biochemical behavior, being prone to aggregation. We also observed that cNEL alone tends to precipitate when concentrated to high concentrations. Consequently, the cROS1/cNEL complex may be suboptimal for rapid freezing and more likely to contact the air–water interface on the cryo-EM grid, making it susceptible to damage by the hydrophobic air-water interface. Given the elongated shape of cROS1, this damage could increase dynamics between the head and leg regions, leaving only the rigid core of cROS1 resolved in the cryo-EM map.\no The density of the second bat-wing of cNEL is less well defined than the protomer that shows clear binding to cROS1. Is it possible that a flexible ROS1 could be bound at the second site? This would be similar to the proposal of lamininG induced clustering (which the authors claim can not be seen directly in the cryoEM because of flexibility) and achieve the same signaling.\nResponse: We thank the reviewer for raising this interesting question. In our cryo-EM analysis, we resolved two distinct 1:2:1 cROS1/cNEL/hNICOL complexes, in which a single ROS1 molecule alternately binds to either cNEL protomer 1 or 2. Our structures further indicate that ROS1 binding stabilizes the conformation of the bound NEL protomer: (1) in conformation 1, protomer 1 is better resolved than protomer 2; (2) in conformation 2, protomer 2 is better resolved than protomer 1.\nIf both NEL protomers were simultaneously bound to ROS1, their cryo-EM densities would be expected to be equally well resolved; however, such particles were not observed in our dataset. We propose that this binding stoichiometry is unlikely due to steric clashes between the two ROS1 molecules when bound to the same 2:1 NEL/NICOL complex. Nevertheless, we cannot rule out the possibility that a 2:1 NEL/NICOL complex could recruit two ROS1 molecules following significant conformational changes in either NEL/NICOL or ROS1. This possibility has been discussed in the revised manuscript.\n6. Ligand species mismatch\no The use of human NICOL with chicken NEL and chicken ROS1 may be problematic. As seen in the supplied alignments, human NICOL contains an additional cysteine that could significantly alter association and activity. The authors should test chicken NICOL in NEL complex to rule out unintended artifacts.\nResponse: We thank the reviewer for raising this important point. To address it, we co-expressed and purified chicken NEL (cNEL) and chicken NICOL (cNICOL). Cell-based activity assays showed that the cNEL/cNICOL co-ligand complex activated ROS1 to a level very similar to that of the cNEL/hNICOL complex (new Supplementary Figure 7). These results suggest that it is unlikely that ligand species mismatch could cause any structural artifacts. As shown in the figure below, the extra cysteine residue in human NICOL (Cys69) is located in the middle part of the helix and does not make any interaction with either NICOL itself or cNEL. This residue is not conserved in other species such as rat, mouse and chicken, suggesting that it is not a functionally important residue.\nMinor Comments\n• The title should specify “chicken ROS1” or “avian ROS1” to avoid overgeneralizing.\nResponse: Point accepted.\n• Replace “apo” with “unliganded” or “ligand-free,” as “apo” is typically used for small-molecule–free enzyme forms.\nResponse: Corrected.\n• Maintain consistent terminology for the unliganded receptor conformation (“extended” vs. “arc-shaped”).\nResponse: Point accepted.\n• The phrase “hNICOL promotes cNEL dimerization” may be misleading, as it implies cNEL is otherwise monomeric. Clarify whether cNEL is trimeric, as expected for other NELLs, and whether NICOL prevents trimerization.\nResponse: Good point. Our SDS-PAGE results showed that cNEL alone exists primarily as a dimer. We also observe small amount of trimers. This is consistent with previous results of NELL1, showing the coexistence of both dimer and trimer (PMID: 24563467, 21814724). We have clarified this point in the revised manuscript.\n• Correct typographical errors: e.g., “cNET/hNICOL” should read “cNEL/hNICOL.”\nResponse: Corrected.\nOverall, the structural data are of high potential interest, but the lack of supporting biochemical and cell-based validation makes it difficult to assess the physiological relevance of the proposed mechanism. Adding quantitative binding studies, oligomeric state analyses, direct ROS1 phosphorylation data, and expanded activation kinetics would substantially strengthen the manuscript.\nResponse: We thank the reviewer for the constructive comments. We hope that the reviewer agree that the additional experiments included the revised manuscript as suggested have substantially strengthened the manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Review of: “Structural Basis for the Activation of the Chicken ROS1 Receptor by the NEL/NICOL Ligand Complex” by An et al.\nThe revised draft adds data and at the same time has toned down some speculation that was without supporting data. Although, I would suggest additional toning down on speculation, and reexamining new data. Below are suggestions that could further clarify points for the readers and make for a more polished manuscript. In particular, Fig 6 could be made more impactful to show the thoughts of the authors in their new Discussion section, where they speculate on the impact of NICOL on activation.\nImportant control (critical)\nFig 2E, 4c and cell activation throughout (Supp 7, Supp 8, Supp 9, Supp 13). There are no controls for ligand alone (cNEL or cNEL/NICOL) tested to parental cells (HEK or H1299) without cROS1 expression. Unless I missed this? Since NEL is known to interact with another receptor ROBO3 (and potentially additional receptors), downstream ERK could come from many sources. This is a necessary control.\nLogic missing regarding the comparisons made between cNEL vs cNEL/NICOL induced activation/mechanism:\nWhat is the proposed mechanism for how NICOL changes signaling of cNEL? Reader is left confused about how NICOL affects function. It is ok to state that it remains yet unknown, and that imparted rigidity may play a factor, and show this as hypothesis in Fig 6. Compared to without NICOL.\nFrom the data: both ligands (NEL dimer and NEL/NICOL heterotrimer) bind equivalently to ROS1, both form higher order oligomers, both have a foundational dimer of cNEL in the complex. Yet robust signaling requires NICOL. The authors essentially present structures in the manuscript of unliganded ROS1 (Fig 1), ligand bound but inhibited (*partly) ROS1 (NEL only) (Fig 2), and ligand bound active ROS1 (NEL+NICOL) (Fig 3). From the visual presentation of figures the logic rather seems: 1) with no ligand ROS1 has a slightly flexible leg region, 2) with cNEL there is more flexibility, whereas 3) with cNEL plus NICOL, ROS1 has a rigidly linked leg region. This, coupled to cNEL’s oligomerization, seems most supported by the structural analysis as a link to function. Authors should mention some of this directly, as the reader will be drawn to these clear structural differences.\nFig 6. Too much attention paid to the speculative (3 different) ways of oligomerization, and this only focuses on with NICOL present. How does NICOL effect compared to what happens without NICOL? This figure should reflect the new Discussion. Fig6 should show what is supported by data: how NEL interacts with ROS1, how this is NEL dimer (with or without NICOL) bound to 1 ROS1, and that some yet to be determined mechanism (not these 3 speculative but similar ways) leads to higher order clustering. With a “?” showing this resolution still not there. Real question throughout manuscript is how NICOL affects activity. This should be compared/contratsed to the minus NICOL cluster model.\nNEL dimer vs trimer\nAuthors state that cNEL is a dimer, with or without NICOL present. This is different than mammalian NELL2 or (NELL1) where there is clear evidence that shows NELLs are trimeric: (Nature Communications 2020, PMID: 32198364). Thus, a dimer is unexpected and not consistent with prior reports. This is interesting, as avian NEL is likely different than mammalian NELLs. Authors should point out this interesting finding.\nIssues with the SECMALLs in Fig 5.\nSome of the masses predicted exceed a megadalton which is not consistent with the elution volume. These peaks are clearly not at the void volume of column (~15min), yet predicted masses for oligomers far exceed size limitation separated by column. Please explain.\nAlso, why did authors only show UV for cNEL/hNICOL? The UV signal shows almost none of the protein to be oligomeric. What about UV traces for others? cNEL alone, seems more significant fraction is oligomer (by LS). For cROS1 what is the shown trace? Is it LS or UV, because LS labeled purple, but curve is yellow.\nWhen one focuses on the linear (non-oligomer) mass predicted of each: it seems cNEL ~300 kDa, cNEL/hNICOL ~300 kDa, and cROS1 ~300 kDa. Is this consistent with authors prediction of dimer cNEL, dimer cNEL+ monomer NICOL, and monomer cROS1? If one then compares these masses to the SECMALs of complexes (cROS1/cNEL with mass ~900kDa, and cROS1/cNEL/hNICOL with mass ~600kDa), this indicates that without NICOL, 2 ROS1’s can actually bind to one cNEL (300+300+300=900). Whereas, with NICOL, only 1 ROS1 can bind (300+300=600). The authors should examine this data for their interpretation. This could fit their structural findings that seem to show increased rigidity with NICOL, and how this might limit a second ROS1 binding event.\nMinor:\nThe title: “Structural basis for the Activation of the Chicken ROS1 Receptor by the NEL/NICOL Ligand Complex” implies that the structural basis for activation has been elucidated. However, the certainty described here is rather for how cNEL engages cROS1. Not for activation per say. The exact structural mode of driving activation through clustering remains to be defined.\nShould Supplemental Figs be called Extended Data?\nThe statements that ROS1 adopts an “extended conformation” is a bit misleading. The N-terminus is in the bent-over conformation as described for Sevenless. “Extended” seems to imply an opening up of this “bent-over” state. The C-terminus ‘leg’ is an extension from this. But one that is not rigid in the absence of ligand. So better described as similar to Sev, N-terminus is folded back, and C-terminal half flexible and dynamic.\nThroughout, the N-term domain referred to as HD for helical domain, but this region has been previously referred to as a CATCH domain for (cysteine-rich ALKAL-type coupled Helices) for the drosophila Sevenless receptor.\nLine 23: “ Structural analyses reveal that the 2:1 NEL/NICOL complex further\noligomerizes through LamG–VWC4 domain interactions, facilitating the clustering of\nmultiple ROS1 for its activation.”\nDoes the authors’ “structural analysis” actually “reveal” this? What structural work unambiguously revealed this interaction?\nOr rather is biochemical experiments suggestive of this?\nLine 77: Despite the present (*presence) of two cNEL protomers in this complex,\nLine 106: “A similar D-shaped fold has been observed in the structure of Sevenless (Sev), the Drosophila ortholog of ROS1, indicating the structural conservation of ROS1 across species.” No citation, or structural comparison made here?\nLine 246: “SEC-MALS and blue native PAGE (BN-PAGE) results showed … cNEL/hNICOL and cROS1/cNEL/hNICOL\ncomplex can further assemble into higher-order oligomers (Fig. 5a,b).”\nBut data also shows cNEL and cNEL-ROS1 (without NICOL) can form same higher order oligomers. Therefore, stating this is in isolation is misleading one to believe that cNEL requires NICOL for additional oligomerization.", "reviewer_2": "As noted previously, The authors present the first full-length extracellular domain structure of a higher vertebrate receptor tyrosine kinase (RTK) ROS1 in it’s apo form and bound to the ligand NEL, and a ligand/co-ligand complex NEL/NICOL. This is an exciting finding that provides new insight into oligomerization via interaction with this co-liganded complex. Activation of RTKs, which are important regulatory signaling molecules and common therapeutic targets, is a topic of great current interest as we are beginning to understand that RTK signaling often has several layers of complexity, and this paper provides some of the first direct structural evidence for clustering mediated by co-ligands, which I think will be of great interest to your readership and something that is widely discussed and cited going forward. Overall, I think the authors have responded adequately to criticisms and questions offered by myself and other reviewers. They provided model and map data that allowed me to answer several of the questions I posed as well. I think the changes to Figure 6 have enhanced the presentations of their conclusions/hypotheses formed by their work. One last comment to the authors: I appreciate their efforts to visualize a receptor:ligand/co-ligand dimer, and just offer one more suggestion to try running ab-initio multiple times where you vary the number of classes (up to 10) to see if they might be able to pull out the dimer just in case this has not been tried. This is not a suggestion within the scope of this publication as they have likely tried this and I think the manuscript already provides significant advances that will be of great interest to numerous readers."}, "author_response": "We thank the reviewer for the positive assessment of our revised manuscript and the suggestions for further improving our manuscript.\n\nImportant control (critical)\nFig 2E, 4c and cell activation throughout (Supp 7, Supp 8, Supp 9, Supp 13). There are no controls for ligand alone (cNEL or cNEL/NICOL) tested to parental cells (HEK or H1299) without cROS1 expression. Unless I missed this? Since NEL is known to interact with another receptor ROBO3 (and potentially additional receptors), downstream ERK could come from many sources. This is a necessary control.\nResponse: Thank you for pointing out this issue. To address this concern, we performed cell-based activation assays in HEK293T cells with or without cROS1 expression. Upon stimulation with cNEL or cNEL/hNICOL, no ERK phosphorylation was detected in HEK293T cells lacking cROS1, whereas robust ERK activation was observed in cells expressing cROS1 (new Supplementary Fig. 7b). These results rule out the involvement of receptors other than ROS1 in mediating cNEL- or cNEL/hNICOL-induced ERK activation.\n\nLogic missing regarding the comparisons made between cNEL vs cNEL/NICOL induced activation/mechanism:\nWhat is the proposed mechanism for how NICOL changes signaling of cNEL? Reader is left confused about how NICOL affects function. It is ok to state that it remains yet unknown, and that imparted rigidity may play a factor, and show this as hypothesis in Fig 6. Compared to without NICOL.\nResponse: We agree with the reviewer that the precise mechanism by which NICOL promotes NEL-dependent ROS1 activation remains unclear. Our structural and biochemical analyses show that NICOL binding stabilizes NEL and improves its biochemical properties. We therefore hypothesize that NICOL enhances the biological activity of NEL by stabilizing its structure and improving its solubility. This point has been incorporated into the revised manuscript: “Our biochemical and cryo-EM data indicate that cNEL exhibits markedly improved biochemical stability and reduced aggregation upon NICOL binding, suggesting that NICOL may act as a chaperon to enhance the biological activity of NEL by stabilizing its structure and improving its solubility. In addition to the chaperone effect, NICOL may facilitate NEL-dependent ROS1 activation through enhancing the formation of the ROS1 active oligomer and positioning multiple ROS1 molecules in a more favorable configuration for activation, although the detail of this mechanism remains unclear.” We have also revised Fig. 6 to reflect this model.\n\nFrom the data: both ligands (NEL dimer and NEL/NICOL heterotrimer) bind equivalently to ROS1, both form higher order oligomers, both have a foundational dimer of cNEL in the complex. Yet robust signaling requires NICOL. The authors essentially present structures in the manuscript of unliganded ROS1 (Fig 1), ligand bound but inhibited (*partly) ROS1 (NEL only) (Fig 2), and ligand bound active ROS1 (NEL+NICOL) (Fig 3). From the visual presentation of figures the logic rather seems: 1) with no ligand ROS1 has a slightly flexible leg region, 2) with cNEL there is more flexibility, whereas 3) with cNEL plus NICOL, ROS1 has a rigidly linked leg region. This, coupled to cNEL’s oligomerization, seems most supported by the structural analysis as a link to function. Authors should mention some of this directly, as the reader will be drawn to these clear structural differences.\nResponse: We thank the reviewer for raising this important point. From a structural perspective, we cannot readily explain how binding of NEL or the NEL/NICOL complex alters the flexibility between the “head” and “leg” regions of ROS1, as the ligand-binding site is located far away from the hinge connecting these two regions, and the conformations of the ROS1 “head” are essentially identical in all three states. We therefore speculate that the observed differences in flexibility of ROS1 may have arisen from cryo-EM sample quality. For example, the ROS1/NEL sample exhibits relatively poor biochemical behavior due to the aggregation tendency of NEL, which may hinder cryo-EM structural determination and result in poorly resolved ROS1 “leg” region.\n\nFig 6. Too much attention paid to the speculative (3 different) ways of oligomerization, and this only focuses on with NICOL present. How does NICOL effect compared to what happens without NICOL? This figure should reflect the new Discussion. Fig6 should show what is supported by data: how NEL interacts with ROS1, how this is NEL dimer (with or without NICOL) bound to 1 ROS1, and that some yet to be determined mechanism (not these 3 speculative but similar ways) leads to higher order clustering. With a “?” showing this resolution still not there. Real question throughout manuscript is how NICOL affects activity. This should be compared/contratsed to the minus NICOL cluster model.\nResponse: We thank the reviewer for the excellent suggestions. In response, we have extensively revised Figure 6 in accordance with the reviewer’s comments in the revised manuscript.\n\nNEL dimer vs trimer\nAuthors state that cNEL is a dimer, with or without NICOL present. This is different than mammalian NELL2 or (NELL1) where there is clear evidence that shows NELLs are trimeric: (Nature Communications 2020, PMID: 32198364). Thus, a dimer is unexpected and not consistent with prior reports. This is interesting, as avian NEL is likely different than mammalian NELLs. Authors should point out this interesting finding.\nResponse: We thank the reviewer for raising this important point. Our purified chicken NEL predominantly forms dimers, but some trimers can be also observed. We notice that in the previous study (Nature Communications 2020, PMID: 32198364), NELL2 was expressed and purified using insect cells, whereas we used a mammalian expression system. Thus, the difference in oligomeric state may arise from either the expression system or species-specific variation, as suggested by the reviewer. This point has been briefly mentioned, and this publication been cited in the revised manuscript: “Interestingly, a previous study indicates that recombinant human NELL1 and NELL2 purified from insect cells predominantly exist as trimers. The observed differences in oligomeric state may therefore arise from differences in the expression system or from species-specific variation.” We have also revised Fig. 6 to indicate the co-existence of dimer and trimer of cNEL alone.\n\nIssues with the SECMALLs in Fig 5.\nSome of the masses predicted exceed a megadalton which is not consistent with the elution volume. These peaks are clearly not at the void volume of column (~15min), yet predicted masses for oligomers far exceed size limitation separated by column. Please explain.\nResponse: Thank you for raising this concern. Although the stated \"optimum\" separation mass range for the column is 10,000-600,000, this does not mean that all masses > 600,000 will be in the void volume. There will be a range between the void volume and the elution volume of the optimal maximum (globular) mass that can be observed but not resolved well.\n\nAlso, why did authors only show UV for cNEL/hNICOL? The UV signal shows almost none of the protein to be oligomeric. What about UV traces for others? cNEL alone, seems more significant fraction is oligomer (by LS). For cROS1 what is the shown trace? Is it LS or UV, because LS labeled purple, but curve is yellow.\nResponse: Good point. We initially included only UV trace for cNEL/hNICOL to emphasize that those species were not present at a high population. We have added the UV traces for other samples in the revised manuscript (new Fig. 5).\nDuring cryo-EM grid screening, we observed that cNEL was prone to aggregation at higher concentrations in the absence of NICOL. Consistent with this observation, SEC showed asymmetric peak tailing, and MALS analysis indicated that a substantial fraction of cNEL existed in higher-order oligomeric states.\nFor cROS1, the trace shown corresponds to the LS. The LS was mistakenly labeled in purple in the previous version. The labeling error has been corrected in the revised manuscript (new Fig. 5). We apologize for this oversight and thank the reviewer for pointing it out.\n\nWhen one focuses on the linear (non-oligomer) mass predicted of each: it seems cNEL ~300 kDa, cNEL/hNICOL ~300 kDa, and cROS1 ~300 kDa. Is this consistent with authors prediction of dimer cNEL, dimer cNEL+ monomer NICOL, and monomer cROS1? If one then compares these masses to the SECMALs of complexes (cROS1/cNEL with mass ~900kDa, and cROS1/cNEL/hNICOL with mass ~600kDa), this indicates that without NICOL, 2 ROS1’s can actually bind to one cNEL (300+300+300=900). Whereas, with NICOL, only 1 ROS1 can bind (300+300=600). The authors should examine this data for their interpretation. This could fit their structural findings that seem to show increased rigidity with NICOL, and how this might limit a second ROS1 binding event.\nResponse: We thank the reviewer for raising this critical point. The theoretical molecular weights of cROS1, cNEL, and hNICOL are 209.2 kDa, 95.6 kDa, and 6.7 kDa, respectively. However, both cNEL and cROS1 are heavily glycosylated. For instance, cROS1 contains approximately 30 glycosylation sites. As a result, the actual molecular weights of cROS1, the cNEL dimer, and the 2:1 cNEL/hNICOL complex may all be close to 300 kDa, which is consistent with the SEC–MALS measurements.\nWe agree with the reviewer that, due to the flexibility of the cNEL dimer, a single cNEL dimer could potentially bind two cROS1 molecules simultaneously. Binding of hNICOL increases the rigidity of the cNEL dimer, thereby preventing association with a second cROS1. We have added the following sentence to the revised manuscript: “Given that cNEL predominantly forms a flexible disulfide-linked dimer, a single cNEL dimer may be capable of binding two cROS1 molecules simultaneously, although such a dimeric complex is not resolved in our cryo-EM analysis.”\n\nMinor:\nThe title: “Structural basis for the Activation of the Chicken ROS1 Receptor by the NEL/NICOL Ligand Complex” implies that the structural basis for activation has been elucidated. However, the certainty described here is rather for how cNEL engages cROS1. Not for activation per say. The exact structural mode of driving activation through clustering remains to be defined.\nResponse: Good point. We have modified the title as “Structural insights into the Activation of the Chicken ROS1 Receptor by the NEL/NICOL Ligand Complex” in the revised manuscripts.\n\nShould Supplemental Figs be called Extended Data?\nResponse: Thank you for your concern. We note that many papers published in Nature Communications label supplementary figures as “Supplementary Figures” rather than “Extended Data,” and we have followed this convention in our manuscript.\n\nThe statements that ROS1 adopts an “extended conformation” is a bit misleading. The N-terminus is in the bent-over conformation as described for Sevenless. “Extended” seems to imply an opening up of this “bent-over” state. The C-terminus ‘leg’ is an extension from this. But one that is not rigid in the absence of ligand. So better described as similar to Sev, N-terminus is folded back, and C-terminal half flexible and dynamic.\nResponse: We thank the reviewer for this helpful comment. We agree that the term “extended conformation” could be misleading. We have therefore changed the “extended conformation” to “arc-shaped conformation” in the revised manuscript.\n\nThroughout, the N-term domain referred to as HD for helical domain, but this region has been previously referred to as a CATCH domain for (cysteine-rich ALKAL-type coupled Helices) for the drosophila Sevenless receptor.\nResponse: Point accepted, we have changed HD domain to CATCH domain in the revised version.\n\nLine 23: “ Structural analyses reveal that the 2:1 NEL/NICOL complex further oligomerizes through LamG–VWC4 domain interactions, facilitating the clustering of multiple ROS1 for its activation.”\nDoes the authors’ “structural analysis” actually “reveal” this? What structural work unambiguously revealed this interaction? Or rather is biochemical experiments suggestive of this?\nResponse: Thank you for the comment. Our cryo-EM density suggests a LamG–VWC2 interaction between two cNEL/hNICOL heterotrimers (Supplementary Fig. 12), which is consistent with biochemical experiments showing that cNEL/hNICOL forms higher-order oligomers and that the LamG domain is essential for oligomer formation (Fig. 5b). However, our cryo-EM reconstruction cannot unambiguously resolve this interaction. Accordingly, we have modified the sentence in the manuscript to: “Structural analyses and biochemical results suggest that the 2:1 NEL/NICOL complexes further oligomerize through LamG–VWC4 domain interactions, facilitating the clustering of multiple ROS1 for its activation.”\n\nLine 77: Despite the present (*presence) of two cNEL protomers in this complex,\nResponse: Corrected.\n\nLine 106: “A similar D-shaped fold has been observed in the structure of Sevenless (Sev), the Drosophila ortholog of ROS1, indicating the structural conservation of ROS1 across species.” No citation, or structural comparison made here?\nResponse: Point accepted. The two papers describing Sevenless structures have now been cited here.\n\nLine 246: “SEC-MALS and blue native PAGE (BN-PAGE) results showed … cNEL/hNICOL and cROS1/cNEL/hNICOL complex can further assemble into higher-order oligomers (Fig. 5a,b).”\nBut data also shows cNEL and cNEL-ROS1 (without NICOL) can form same higher order oligomers. Therefore, stating this is in isolation is misleading one to believe that cNEL requires NICOL for additional oligomerization.\nResponse: Good point. We have revised this sentence: “Indeed, both SEC-MALS and blue native PAGE (BN-PAGE) results showed that cROS1 alone predominantly exists as a monomer, whereas the cNEL, cNEL/hNICOL, cROS1/cNEL and cROS1/cNEL/hNICOL complex can further assemble into higher-order oligomers (Fig. 5a, b).”."}, {"round": 3, "reviewer_comments": {"reviewer_1": "======================================================================\nATTACHED REVIEWER REPORT — IMPORTANT\nThe text below is a complete reviewer report submitted as a PDF\nattachment. It appears after the Creative Commons license block.\nAssign it to whichever reviewer wrote 'see pdf' or had no inline text.\n======================================================================\n\nReviewer #1 (Remarks to the Author):\nThis is an interesting and timely study that advances our understanding of vertebrate (chicken) ROS1\nregulation. To date, only Drosophila ROS1 (Sevenless) has been structurally and mechanistically\ncharacterized, and it employs an entirely different ligand and activation mechanism that is not\nconserved in vertebrates. Here, the authors use chicken ROS1 (cROS1) and its ligand NEL, with or\nwithout the co-ligand NICOL, to determine structures that point towards a novel mode of regulation.\nThe cryo-EM work appears to be of high quality and at resolutions sufficient to define observed\ninteractions. These findings represent an important step toward understanding the molecular basis of\nvertebrate ROS1 signaling.\nWe thank the reviewer for the positive assessment of our manuscript.\nHowever, several major issues need to be addressed before the conclusions can be fully supported.\nMajor Concerns\n1. Supporting biophysical characterization\no While the structures are well executed, there is no quantitative binding analysis (SPR/BLI)\ncomparing ROS1–NEL and ROS1–NEL/NICOL interactions.\nReviewer #2 (Remarks to the Author):\nThe authors present the first full-length extracellular domain structure of a higher vertebrate receptor\ntyrosine kinase (RTK) ROS1 in it’s apo form and bound to the ligand NEL, and a ligand/co-ligand complex\nNEL/NICOL. This is an exciting finding that provides new insight into oligomerization via interaction with\nthis co-liganded complex. Activation of RTKs, which are important regulatory signaling molecules and\ncommon therapeutic targets, is a topic of great current interest as we are beginning to understand that\nRTK signaling often has several layers of complexity, and this paper provides some of the first direct\nstructural evidence for clustering mediated by co-ligands, which I think will be of great interest to your\nreadership and something that is widely discussed and cited going forward.\nThe authors provide a careful analysis of the three structures and follow up key findings with functional\nassays. It’s also interesting that sugars are clear mediators of some domain interactions in apo ROS1,\nhighlighting the importance of determining these structures with glycans included, as the authors did\nhere. This is worth mentioning, because historically many RTK structures are deglycosylated, a practice\nwhich has clearly hidden important features of structural organization. I’ve included a few comments\nbelow that I thought were important to make:\nReviewer #3 (Remarks to the Author):\nOverall Assessment\nThis manuscript reports the cryo-EM structure of the NEL–NICOL–ROS1 complex, providing high-\nresolution structural insight into ROS1 activation by its recently identified endogenous ligands, NELL2\nand NICOL. While ROS1 has long been recognized in oncology as a receptor tyrosine kinase activated by\noncogenic fusion proteins, the physiological ligands and activation mechanisms have remained unclear\nuntil recent work identified the NELL2/NICOL complex as a key mediator of testis-to-epididymis signaling\nand male fertility. From a structural biology perspective, no detailed architecture of this ternary complex\nhas been described, making the present work timely and potentially significant.\nThe study’s key strength lies in the novelty of the structural findings, which extend our understanding of\nROS1 signaling beyond the oncogenic context and provide a foundation for exploring distinctive modes\nof RTK activation. The cryo-EM data reveal a ligand–co-ligand–receptor arrangement that offers a\nstructural framework likely to inspire new hypotheses about receptor clustering and signal initiation.\nThe topic will interest structural biologists, reproductive biologists, and the broader RTK signaling\ncommunity.\nReviewer #1: \nThe authors addressed all of my concerns", "reviewer_2": "Reviewer #1: \nThe authors addressed all of my concerns"}, "author_response": "Reviewer #1: \nThe authors addressed all of my concerns\nResponse: we thank the reviewer for supporting the publication.\n\nReviewer #2 (Remarks to the Author):\nAs noted previously, The authors present the first full-length extracellular domain structure of a higher vertebrate receptor tyrosine kinase (RTK) ROS1 in it’s apo form and bound to the ligand NEL, and a ligand/co-ligand complex NEL/NICOL. This is an exciting finding that provides new insight into oligomerization via interaction with this co-liganded complex. Activation of RTKs, which are important regulatory signaling molecules and common therapeutic targets, is a topic of great current interest as we are beginning to understand that RTK signaling often has several layers of complexity, and this paper provides some of the first direct structural evidence for clustering mediated by co-ligands, which I think will be of great interest to your readership and something that is widely discussed and cited going forward. Overall, I think the authors have responded adequately to criticisms and questions offered by myself and other reviewers. They provided model and map data that allowed me to answer several of the questions I posed as well. I think the changes to Figure 6 have enhanced the presentations of their conclusions/hypotheses formed by their work. One last comment to the authors: I appreciate their efforts to visualize a receptor:ligand/co-ligand dimer, and just offer one more suggestion to try running ab-initio multiple times where you vary the number of classes (up to 10) to see if they might be able to pull out the dimer just in case this has not been tried. This is not a suggestion within the scope of this publication as they have likely tried this and I think the manuscript already provides significant advances that will be of great interest to numerous readers.\nResponse: Thank you for the positive assessment of our revised manuscript and for the helpful suggestions. We explored multiple strategies for ab initio reconstruction, including the approach suggested by the reviewer (varying the number of classes). Despite these efforts, we were unable to resolve a larger complex, likely due to the intrinsic flexibility of this higher-order assembly. We believe that biochemical stabilization of the higher-order ROS/NEL/NICOL complex, or improved sample preparation (for example, using full-length ROS1 for structural studies), will be essential for capturing its structure and will ultimately provide a more complete understanding of how ROS1 is activated by NEL and NICOL."}]

Decision letter
Peer Review File
Structural Insights into the Activation of the Chicken ROS1
Receptor by the NEL/NICOL Ligand Complex
Corresponding Author: Dr Xiaochen Bai
This file contains all reviewer reports in order by version, followed by all author rebuttals in order by version.